Compositions and methods for silencing apolipoprotein b

ABSTRACT

The present invention provides compositions and methods for the delivery of interfering RNAs that silence APOB expression to liver cells. In particular, the nucleic acid-lipid particles provide efficient encapsulation of nucleic acids and efficient delivery of the encapsulated nucleic acid to cells in vivo. The compositions of the present invention are highly potent, thereby allowing effective knock-down of APOB at relatively low doses. In addition, the compositions and methods of the present invention are less toxic and provide a greater therapeutic index compared to compositions and methods previously known in the art.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 61/222,464, filed Jul. 1, 2009, and U.S. Provisional Application No.61/351,275, filed Jun. 3, 2010, the disclosures of which are herebyincorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

Apolipoprotein B (also known as ApoB, apolipoprotein B-100; ApoB-100,apolipoprotein B-48; ApoB-48 and Ag(x) antigen), is a large glycoproteinthat serves an indispensable role in the assembly and secretion oflipids and in the transport and receptor-mediated uptake and delivery ofdistinct classes of lipoproteins. Apolipoprotein B was cloned (Law etal., PNAS USA 82:8340-8344 (1985)) and mapped to chromosome 2p23-2p24 in1986 (Deeb et al., PNAS USA 83, 419-422 (1986)). ApoB has a variety offunctions, from the absorption and processing of dietary lipids to theregulation of circulating lipoprotein levels (Davidson and Shelness,Annu. Rev. Nutr., 20:169-193 (2000)). Two forms of ApoB have beencharacterized: ApoB-100 and ApoB-48. ApoB-100 is the major proteincomponent of LDL, contains the domain required for interaction of thislipoprotein species with the LDL receptor, and participates in thetransport and delivery of endogenous plasma cholesterol (Davidson andShelness, 2000, supra). ApoB-48 circulates in association withchylomicrons and chylomicron remnants which are cleared by theLDL-receptor-related protein (Davidson and Shelness, 2000, supra).ApoB-48 plays a role in the delivery of dietary lipid from the smallintestine to the liver.

Susceptibility to atherosclerosis is highly correlated with the ambientconcentration of apolipoprotein B-containing lipoproteins (Davidson andShelness, 2000, supra). Elevated plasma levels of theApoB-100-containing lipoprotein Lp(a) are associated with increased riskfor atherosclerosis and its manifestations, which may includehypercholesterolemia (Seed et al., N. Engl. J. Med. 322:1494-1499(1990), myocardial infarction (Sandkamp et al., Clin. Chem. 36:20-23(1990), and thrombosis (Nowak-Gottl et al., Pediatrics, 99:E11 (1997)).

Apolipoprotein B knockout mice (bearing disruptions of both ApoB-100 andApoB-48) have been generated which are protected from developinghypercholesterolemia when fed a high-fat diet (Farese et al., PNAS USA.92:1774-1778 (1995) and Kim and Young, J. Lipid Res., 39:703-723(1998)). The incidence of atherosclerosis has been investigated in miceexpressing exclusively ApoB-100 or ApoB-48 and susceptibility toatherosclerosis was found to be dependent on total cholesterol levels.

In view of such findings, significant efforts have been made to modulateserum cholesterol levels by modulating ApoB expression using therapeuticnucleic acids, e.g., antisense oligonucleotides, ribozymes, etc. (see,e.g., U.S. Pat. No. 7,407,943, which is directed to modulation of ApoBusing antisense oligonucleotides). More recent efforts have focused onthe use of interfering RNA molecules, such as siRNA and miRNA, tomodulate ApoB (see, Zimmermann et al., Nature, 441: 111-114 (2006), U.S.Patent Publication Nos. 20060134189 and 20060105976, and PCT PublicationNo. WO 04/091515). Interfering RNA molecules can down-regulateintracellular levels of specific proteins, such as ApoB, through aprocess termed RNA interference (RNAi). Following introduction ofinterfering RNA into the cell cytoplasm, these double-stranded RNAconstructs can bind to a protein termed RISC. The sense strand of theinterfering RNA is displaced from the RISC complex, providing a templatewithin RISC that can recognize and bind mRNA with a complementarysequence to that of the bound interfering RNA. Having bound thecomplementary mRNA, the RISC complex cleaves the mRNA and releases thecleaved strands. RNAi can provide down-regulation of specific proteins,such as ApoB, by targeting specific destruction of the correspondingmRNA that encodes for protein synthesis.

Despite the high therapeutic potential of RNAi, two problems currentlyfaced by interfering RNA constructs are, first, their susceptibility tonuclease digestion in plasma and, second, their limited ability to gainaccess to the intracellular compartment where they can bind RISC whenadministered systemically as free interfering RNA molecules. Thesedouble-stranded constructs can be stabilized by the incorporation ofchemically modified nucleotide linkers within the molecule, e.g.,phosphothioate groups. However, such chemically modified linkers provideonly limited protection from nuclease digestion and may decrease theactivity of the construct.

In an attempt to improve efficacy, investigators have employed variouslipid-based carrier systems to deliver chemically modified or unmodifiedtherapeutic nucleic acids, including anionic (conventional) liposomes,pH sensitive liposomes, immunoliposomes, fusogenic liposomes, andcationic lipid/nucleic acid aggregates. In particular, one lipid-basedcarrier system, i.e., the stable nucleic-acid lipid particle (SNALP)system, has been found to be particularly effective for deliveringinterfering RNA (see, U.S. Patent Publication No. 20050064595 and U.S.Patent Publication No. 20060008910 (collectively referred to as“MacLachlan et al.”)). MacLachlan et al. have demonstrated thatinterfering RNA, such as siRNA, can be effectively systemicallyadministered using nucleic acid-lipid particles containing a cationiclipid, and that these nucleic acid-lipid particles provide improveddown-regulation of target proteins in mammals including non-humanprimates (see, Zimmermann et al., Nature, 441: 111-114 (2006)).

Even in spite of this progress, there remains a need in the art forimproved SNALPs that are useful for delivering therapeutic nucleicacids, such as siRNA and miRNA, to the liver of a mammal (e.g., ahuman), and that result in increased silencing of target genes ofinterest in the liver, such as ApoB. Preferably, these compositionswould encapsulate nucleic acids with high-efficiency, have highdrug:lipid ratios, protect the encapsulated nucleic acid fromdegradation and clearance in serum, be suitable for systemic delivery,and provide intracellular delivery of the encapsulated nucleic acid. Inaddition, these nucleic acid-lipid particles should be well-toleratedand provide an adequate therapeutic index, such that patient treatmentat an effective dose of the nucleic acid is not associated withsignificant toxicity and/or risk to the patient. The present inventionprovides such compositions, methods of making the compositions, andmethods of using the compositions to introduce nucleic acids, such assiRNA and miRNA, into the liver, including for the treatment ofdiseases, such as hypercholesterolemia (e.g., atherosclerosis, anginapectoris or high blood pressure).

BRIEF SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery that the useof certain cationic (amino) lipids in nucleic acid-lipid particlesprovides advantages when the particles are used for the in vivo deliveryof therapeutic nucleic acids, such as siRNA, into the liver of a mammal.In particular, it has been unexpectedly found that the nucleicacid-lipid particles of the present invention comprising at least onecationic lipid of Formula I-XIV and at least one interfering RNA asdescribed herein demonstrate increased potency (i.e., increasedsilencing activity) and/or increased tolerability (e.g., a morefavorable toxicity profile) when targeting a gene of interest in theliver such as APOB, APOC3, PCSK9, DGAT1, and/or DGAT2 when compared toother nucleic acid-lipid particle compositions previously described. Inpreferred embodiments, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) comprising APOB siRNA 3/5 and the cationic lipidDLin-K-C2-DMA and methods of use thereof, which nucleic acid-lipidparticles unexpectedly possess increased potency and increasedtolerability when silencing APOB expression in vivo compared to othernucleic acid-lipid particle compositions previously described.

In particular embodiments, the present invention provides cationiclipids that enable the formulation of compositions for the in vitro andin vivo delivery of interfering RNA, such as siRNA, to the liver thatresult in increased silencing of the target gene of interest, such asAPOB. It is shown herein that these improved lipid particle compositionsare particularly effective in down-regulating (e.g., silencing) theprotein levels and/or mRNA levels of target genes in the liver, such asAPOB. Furthermore, it is shown herein that the activity of theseimproved lipid particle compositions is dependent on the presence of thecationic lipids of Formula I-XIV of the invention.

In one aspect, the present invention provides a nucleic acid-lipidparticle (e.g., SNALP) comprising:

(a) an interfering RNA that silences Apolipoprotein B (APOB) expressionand/or the expression of another liver target gene such as APOC3, PCSK9,DGAT1, and/or DGAT2;

(b) a cationic lipid of Formula I having the following structure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently optionally substituted C₁₂-C₂₄ alkyl, optionallysubstituted C₁₂-C₂₄ alkenyl, optionally substituted C₁₂-C₂₄ alkynyl, oroptionally substituted C₁₂-C₂₄ acyl, with the proviso that at least oneof R¹ and R² has at least two sites of unsaturation; R³ and R⁴ areeither the same or different and are independently optionallysubstituted C₁-C₆ alkyl, optionally substituted C₂-C₆ alkenyl, oroptionally substituted C₂-C₆ alkynyl or R³ and R⁴ may join to form anoptionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or2 heteroatoms chosen from nitrogen and oxygen; R⁵ is either absent orhydrogen or C₁-C₆ alkyl to provide a quaternary amine; m, n and p areeither the same or different and are independently either 0, 1 or 2,with the proviso that m, n, and p are not simultaneously 0; q is 0, 1,2, 3, or 4; Y and Z are either the same or different and areindependently O, S, or NH; and

(c) a non-cationic lipid.

In another aspect, the present invention provides a nucleic acid-lipidparticle (e.g., SNALP) comprising:

(a) an interfering RNA that silences Apolipoprotein B (APOB) expressionand/or the expression of another liver target gene such as APOC3, PCSK9,DGAT1, and/or DGAT2;

(b) a cationic lipid of Formula II having the following structure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently optionally substituted C₁₂-C₂₄ alkyl, optionallysubstituted C₁₂-C₂₄ alkenyl, optionally substituted C₁₂-C₂₄ alkynyl, oroptionally substituted C₁₂-C₂₄ acyl; R³ and R⁴ are either the same ordifferent and are independently optionally substituted C₁-C₆ alkyl,optionally substituted C₂-C₆ alkenyl, or optionally substituted C₂-C₆alkynyl or R³ and R⁴ may join to form an optionally substitutedheterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosenfrom nitrogen and oxygen; R⁵ is either absent or is hydrogen or C₁-C₆alkyl to provide a quaternary amine; m, n, and p are either the same ordifferent and are independently either 0, 1 or 2, with the proviso thatm, n, and p are not simultaneously 0; Y and Z are either the same ordifferent and are independently O, S, or NH; and

(c) a non-cationic lipid.

In some embodiments, cationic lipids falling within the scope ofFormulas I and/or II that are useful in the nucleic acid-lipid particlesof the present invention include, but are not limited to, the following:2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2” or “C2K”),2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA;“C3K”), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane(DLin-K-C4-DMA; “C4K”),2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA),2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DO-K-DMA),2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DS-K-DMA),2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane (DLin-K-MA),2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride(DLin-K-TMA.Cl),2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)[1,3]-dioxolane(DLin-K²-DMA), 2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane(D-Lin-K-N-methylpiperzine), analogs thereof, salts thereof, andmixtures thereof.

In yet another aspect, the present invention provides a nucleicacid-lipid particle (e.g., SNALP) comprising: (a) an interfering RNAthat silences Apolipoprotein B (APOB) expression and/or the expressionof another liver target gene such as APOC3, PCSK9, DGAT1, DGAT2, etc.);(b) a cationic lipid having the structure of Formula III-XIV; and (c) anon-cationic lipid. Examples of cationic lipids falling within the scopeof Formula III-XIV that are useful in the nucleic acid-lipid particlesof the present invention include, but are not limited to, the following:1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA),γ-DLen-C2K-DMA, DLen-C2K-DMA, DPan-C2K-DMA, DPan-C3K-DMA,1,2-dilinoleyloxy-3-piperidinopropylamine (DLinPip),1,2-dilinoleyloxy-3-(3′-hydroxypiperidino)-propylamine (DLinPip-3OH),1,2-dilinoleyloxy-3-(4′-hydroxypiperidino)-propylamine (DLinPip-4OH),1,2-dilinoleyloxy-3-(N,N dimethyl)-propylamine (DLinDEA),N1-((2,3-linoleyloxy)propyl)-N1,N3,N3-trimethylpropane-1,3-diamine(2N-DLinDMA), 1,2-Dilinoleyloxy-3-(1-imidazole)propylamine (DLinIm),1,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDMA),1,2-diphytanyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DPanDMA),1,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDAP),Linoleyl/Oleyl DMA, Linoleyl/Phytanyl DMA, Linoleyl/Linolenyl DMA,Linoleyl/Stearyl DMA, Linoleyl/C₆:0 DMA, Linoleyl/C₆:1 DMA,1-(2,3-linoleyloxypropoxy)-2-(linoleyloxy)-(N,N-dimethyl)-propyl-3-amine(TLinDMA), C2-TLinDMA, DHep-C2K-DMA, DLin-C2K-Pip-3OH,1,2-diarachidonyloxy-(N,N-dimethyl)-propyl-3-amine (DAraDMA),1,2-didocosahexaenyloxy-(N,N-dimethyl)-propyl-3-amine (DDocDMA),1,2-diphytanyloxy-3-(N,N-dimethyl)-propylamine (DPanDMA), 6-memberedketal lipids such as DPan-C1K6-DMA, analogs thereof, salts thereof, andmixtures thereof.

In some embodiments, the lipid particles of the invention preferablycomprise an interfering RNA that silences APOB and/or other liver targetgenes such as APOC3, PCSK9, DGAT1, DGAT2, or combinations thereof, acationic lipid of Formula I-XIV as disclosed herein, a non-cationiclipid, and a conjugated lipid that inhibits aggregation of particles.

In certain embodiments, the non-cationic lipid component of the lipidparticle may comprise a phospholipid, cholesterol (or cholesterolderivative), or a mixture thereof. In one particular embodiment, thephospholipid comprises dipalmitoylphosphatidylcholine (DPPC),distearoylphosphatidylcholine (DSPC), or a mixture thereof. In someembodiments, the conjugated lipid component of the lipid particlecomprises a polyethyleneglycol (PEG)-lipid conjugate. In certaininstances, the PEG-lipid conjugate comprises a PEG-diacylglycerol(PEG-DAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or amixture thereof.

In some embodiments, the interfering RNA is fully encapsulated withinthe lipid portion of the lipid particle such that the interfering RNA inthe lipid particle is resistant in aqueous solution to enzymaticdegradation, e.g., by a nuclease or protease. Non-limiting examples ofinterfering RNA include siRNA, aiRNA, miRNA, Dicer-substrate dsRNA,shRNA, and mixtures thereof. In other embodiments, the lipid particlesdescribed herein are substantially non-toxic to mammals such as humans.

In other embodiments, the nucleic acid-lipid particle comprises aninterfering RNA (e.g., siRNA) that targets APOB, wherein the interferingRNA comprises an antisense strand comprising the sequence5′-UAUUCAGUGUGAUGACACU-3′. In still other embodiments, the nucleic,acid-lipid particle further comprises a sense strand comprising thesequence 5′-AGUGUCAUCACACUGAAUA-3′. In certain embodiments, theinterfering RNA comprises a 3′ overhang in one or both strands of theinterfering RNA molecule. In certain embodiments, the interfering RNAcomprises an antisense strand comprising a 5′-UG-3′ overhang and/or asense strand comprising a 5′-CC-3′ overhang.

In yet other embodiments, the nucleic acid-lipid particle comprises aninterfering RNA (e.g., siRNA) that targets APOB, wherein the interferingRNA comprises at least one modified nucleotide. In certain embodiments,one or more of the nucleotides in the double-stranded region of theinterfering RNA comprise modified nucleotides. In certain otherembodiments, one or more of the nucleotides in the 3′ overhang in one orboth strands of the interfering RNA comprise modified nucleotides. Inparticular embodiments; the modified nucleotides comprise 2′-O-methyl(2′OMe) nucleotides.

In further embodiments, the nucleic acid-lipid particle comprises aninterfering RNA (e.g., siRNA) that targets APOB, wherein the interferingRNA comprises an antisense strand comprising the sequence5′-UAUUCAGUGUGAUGACACU-3′, wherein the bolded and underlined nucleotidesare 2′OMe nucleotides. In other embodiments, the particle furthercomprises a sense strand comprising the sequence5′-AGUGUCAUCACACUGAAUA-3′, wherein the bolded and underlined nucleotidesare 2′OMe nucleotides. In certain embodiments, the interfering RNAcomprises a 3′ overhang in one or both strands of the interfering RNAmolecule. In some embodiments, the interfering RNA comprises anantisense strand comprising a 5′-UG-3′ overhang and/or a sense strandcomprising a 5′-CC-3′ overhang, wherein the bolded and underlinednucleotides are 2′OMe nucleotides. In other embodiments, the nucleicacid-lipid particle comprises an interfering RNA consisting of thefollowing sequences:

5′-AG U G U CA U CACAC UG AA U ACC-3′ and 3′- GUU CACAG U AG U G U GAC UUAU-5′wherein the bolded and underlined nucleotides are 2′OMe nucleotides.

The present invention also provides pharmaceutical compositionscomprising a nucleic acid-lipid particle described herein (e.g., SNALP)and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides methods forintroducing one or more interfering RNA molecules (e.g., siRNAs thatsilence APOB expression and/or the expression of other liver targetgenes such as APOC3, PCSK9, DGAT1, and/or DGAT2) into a cell (e.g., aliver cell), the method comprising contacting the cell with a nucleicacid-lipid particle described herein (e.g., SNALP). In one embodiment,the cell is in a mammal and the mammal is a human.

In yet another aspect, the present invention provides methods for the invivo delivery of one or more interfering RNA molecules (e.g., siRNAs) toliver cells, the method comprising administering to a mammal a nucleicacid-lipid particle described herein (e.g., SNALP). Advantageously, thenucleic acid-lipid particles of the invention are particularly effectiveat silencing target gene expression in the liver and, thus, are wellsuited for targeting genes such as APOB, APOC3, PCSK9, DGAT1, DGAT2, andcombinations thereof. In certain embodiments, the nucleic acid-lipidparticles (e.g., SNALP) are administered by one of the following routesof administration: oral, intranasal, intravenous, intraperitoneal,intramuscular, intra-articular, intralesional, intratracheal,subcutaneous, and intradermal. In particular embodiments, the nucleicacid-lipid particles (e.g., SNALP) are administered systemically, e.g.,via enteral or parenteral routes of administration. In preferredembodiments, the mammal is a human.

In certain embodiments, the present invention provides methods fortreating a liver disease or disorder by administering an interfering RNA(e.g., one or more siRNAs targeting APOB, APOC3, PCSK9, DGAT1, and/orDGAT2 expression) in nucleic acid-lipid particles (e.g., SNALP) asdescribed herein, alone or in combination with a lipid-lowering agent.Examples of lipid diseases and disorders include, but are not limitedto, dyslipidemia (e.g., hyperlipidemias such as elevated triglyceridelevels (hypertriglyceridemia) and/or elevated cholesterol levels(hypercholesterolemia)), atherosclerosis, coronary heart disease,coronary artery disease, atherosclerotic cardiovascular disease (CVD),fatty liver disease (hepatic steatosis), abnormal lipid metabolism,abnormal cholesterol metabolism, diabetes (including Type 2 diabetes),obesity, cardiovascular disease, and other disorders relating toabnormal metabolism. Non-limiting examples of lipid-lowering agentsinclude statins, fibrates, ezetimibe, thiazolidinediones, niacin,beta-blockers, nitroglycerin, calcium antagonists, and fish oil.

In one particular embodiment, the present invention provides a methodfor lowering or reducing cholesterol levels in a mammal (e.g., human) inneed thereof (e.g., a mammal with elevated blood cholesterol levels),the method comprising administering to the mammal a therapeuticallyeffective amount of a nucleic acid-lipid particle (e.g., a SNALPformulation) described herein comprising one or more interfering RNAs(e.g., siRNAs) that target one or more genes associated with metabolicdiseases and disorders (e.g., APOB, APOC3, PCSK9, DGAT1, and/or DGAT2).In another particular embodiment, the present invention provides amethod for lowering or reducing triglyceride levels in a mammal (e.g.,human) in need thereof (e.g., a mammal with elevated blood triglyceridelevels), the method comprising administering to the mammal atherapeutically effective amount of a nucleic acid-lipid particle (e.g.,a SNALP formulation) described herein comprising one or more interferingRNAs (e.g., siRNAs) that target one or more genes associated withmetabolic diseases and disorders (e.g., APOB, APOC3, PCSK9, DGAT1,and/or DGAT2). These methods can be carried out in vitro using standardtissue culture techniques or in vivo by administering the interferingRNA (e.g., siRNA) using any means known in the art. In preferredembodiments, the interfering RNA (e.g., siRNA) is delivered to a livercell (e.g., hepatocyte) in a mammal such as a human.

Additional embodiments related to treating a liver disease or disorderusing a lipid particle are described in, e.g., PCT Application No.PCT/CA2010/000120, filed Jan. 26, 2010, and U.S. Patent Publication No.20060134189, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

In a further aspect, the present invention provides methods for treatinga disease or disorder associated with overexpression of APOB in a mammal(e.g., human) in need thereof, the method comprising administering tothe mammal a therapeutically effective amount of a nucleic acid-lipidparticle (e.g., SNALP) comprising one (or more) interfering RNA thatsilences APOB expression. Diseases and disorders associated withoverexpression of APOB include, but are not limited to, atherosclerosis,angina pectoris, high blood pressure, diabetes, and hypothyroidism. Inpreferred embodiments, the mammal (e.g., human) has a disease ordisorder involving hypercholesterolemia and serum cholesterol levels arelowered when expression of APOB is silenced by the interfering RNAdelivered using the nucleic acid-lipid particles of the presentinvention.

The nucleic acid-lipid particles of the invention (e.g., SNALP)comprising one or more cationic lipids of Formula I-XIV or salts thereofare particularly advantageous and suitable for use in the administrationof interfering RNA to a subject (e.g., a mammal such as a human) becausethey are stable in circulation, of a size required for pharmacodynamicbehavior resulting in access to extravascular sites, and are capable ofreaching target cell populations.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a comparison of the plasma total cholesterol knockdownefficacy of exemplary APOB SNALP formulations containing variouscationic lipids described herein.

FIG. 2 shows a comparison of the liver ApoB mRNA knockdown activity ofexemplary APOB SNALP formulations containing various cationic lipidsdescribed herein.

FIG. 3 shows a comparison of the liver ApoB mRNA knockdown activity ofadditional exemplary APOB SNALP formulations containing various cationiclipids described herein.

FIG. 4 shows a dose response evaluation of three different doses ofexemplary APOB SNALP formulations containing various cationic lipidsdescribed herein on liver ApoB mRNA knockdown activity.

FIG. 5 shows a comparison of the liver ApoB mRNA knockdown activity ofadditional exemplary APOB SNALP formulations containing various cationiclipids described herein.

FIG. 6 shows a comparison of the liver ApoB mRNA knockdown activity ofadditional exemplary APOB SNALP formulations containing various cationiclipids described herein.

FIG. 7 shows a comparison of the liver ApoB mRNA knockdown activity ofadditional exemplary APOB SNALP formulations containing various cationiclipids described herein.

FIG. 8 shows a dose response evaluation of three different doses ofexemplary APOB SNALP formulations containing either DLinDMA or C2K onliver ApoB mRNA knockdown activity.

FIG. 9 shows the reproducibility of liver ApoB mRNA knockdown using twoindependent SNALP batches.

FIG. 10 shows a comparison of the liver ApoB mRNA knockdown activity ofexemplary APOB SNALP formulations containing either DLinDMA or C2K inrats.

FIG. 11 shows a comparison of the TNF inflammatory response in donors toexemplary APOB SNALP formulations containing either DLinDMA or C2K.

FIG. 12 shows a comparison of the IL-8 inflammatory response in donorsto exemplary APOB SNALP formulations containing either DLinDMA or C2K.

FIG. 13 shows a comparison of APOB mRNA knockdown activity of exemplary2′OMe-modified APOB SNALP formulations containing C2K in human primaryhepatocytes.

FIG. 14 shows a comparison of APOB mRNA knockdown activity of exemplary2′OMe-modified APOB SNALP formulations containing either DLinDMA or C2Kin human primary hepatocytes.

FIG. 15 shows a comparison of the liver ApoB mRNA knockdown activity ofexemplary 2′OMe-modified APOB SNALP formulations containing C2K in mice.

FIG. 16 shows a comparison of the liver ApoB mRNA knockdown activity ofexemplary 2′OMe-modified APOB SNALP formulations containing eitherDLinDMA or C2K in mice.

FIG. 17 shows a dose response evaluation of three different doses ofexemplary APOB SNALP formulations containing either DLinDMA or C2K andeither APOB siRNA 1/1 (“siApoB-8”) or APOB siRNA 3/5 (“siApoB-10”) onliver ApoB mRNA knockdown activity.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based, in part, on the discovery that the useof certain cationic (amino) lipids in nucleic acid-lipid particlesprovide advantages when the particles are used for the in vivo deliveryof therapeutic nucleic acids, such as siRNA, into the liver of a mammal.In particular, it has been unexpectedly found that the nucleicacid-lipid particles of the present invention (i.e., SNALP formulations)containing at least one cationic lipid of Formula I-XIV and at least oneinterfering RNA (e.g., siRNA) as described herein demonstrate increasedpotency (i.e., increased silencing activity) and/or increasedtolerability (e.g., a more favorable toxicity profile) when targeting agene of interest in the liver, such as APOB, when compared to othernucleic acid-lipid particle compositions previously described.

In particular embodiments, the present invention provides cationiclipids that enable the formulation of compositions for the in vitro andin vivo delivery of interfering RNA, such as siRNA, to the liver thatresult in increased silencing of the target gene of interest in theliver. It is shown herein that these improved lipid particlecompositions are particularly effective in down-regulating (e.g.,silencing) the protein levels and/or mRNA levels of target genes in theliver, such as APOB. Furthermore, it is shown herein that the activityof these improved lipid particle compositions is dependent on thepresence of the cationic lipids of the invention.

The lipid particles and compositions of the present invention may beused for a variety of purposes, including the delivery of encapsulatedinterfering RNA, such as siRNA, to liver cells (e.g., hepatocytes), bothin vitro and in vivo. Accordingly, the present invention furtherprovides methods of treating metabolic diseases or disorders in asubject in need thereof by contacting the subject with a lipid particlethat encapsulates or is associated with a suitable therapeutic agent,wherein the lipid particle comprises one or more of the novel cationiclipids described herein.

In particular, the lipid particles and compositions of the presentinvention are useful for silencing APOB expression to treat diseases ordisorders associated with expression or overexpression of APOB. Suchdiseases include, e.g., atherosclerosis, angina pectoris, high bloodpressure, diabetes, hypothyroidism, and hypercholesterolemia. In view oftheir enhanced potency, the nucleic acid-lipid particles of the presentinvention comprising an siRNA sequence that targets APOB can effectivelybe used to lower serum cholesterol levels.

As described herein, the lipid particles of the present invention havebeen found to provide more potent silencing when used to deliverinterfering RNA molecules, such as siRNA, to the liver, when compared tolipid particle compositions previously described. As such, in additionto being useful for silencing APOB, the lipid particles of the presentinvention are also use for targeting other genes of interest in theliver. Such genes of interest include, but are not limited to, APOC3,PCSK9, DGAT1, DGAT2, and combinations thereof.

As explained herein, it has surprisingly been found that the lipidparticles of the present invention (e.g., SNALP) containing at least onecationic lipid of Formulas I-XIV, either alone or in combination withother cationic lipids, show increased potency and/or increasedtolerability when targeting a gene of interest in the liver, such as,e.g., APOB, APOC3, PCSK9, DGAT1, and/or DGAT2, when compared to otherSNALP formulations. For instance, as set forth in the Examples below, ithas been found that a lipid particle (e.g., SNALP) containing, e.g.,DLin-K-C2-DMA (“C2K”), γ-DLenDMA, Linoleyl/Linolenyl DMA (“Lin/Len”),C2-DPanDMA, DPan-C2K-DMA, DPan-C3K-DMA, γ-DLen-C2K-DMA, DLen-C2K-DMA, orC2-TLinDMA was unexpectedly more potent in silencing APOB expression invivo compared to SNALP containing DLinDMA or DLenDMA. In addition, asset forth in the Examples below, it has been found that a lipid particle(e.g., SNALP) comprising an APOB siRNA described herein and containing,e.g., DLin-K-C2-DMA, displayed an unexpectedly more favorable toxicityprofile in vivo compared to SNALP formulations containing DLinDMA. Assuch, in certain preferred embodiments, the lipid particles of thepresent invention (e.g., SNALP) comprise a 1:57, 1:62, 7:54, or 7:58lipid particle (e.g., SNALP) containing one or more cationic lipids ofFormulas I-XIV, such as C2K, γ-DLenDMA, Linoleyl/Linolenyl DMA(“Lin/Len”), C2-DPanDMA, DPan-C2K-DMA, DPan-C3K-DMA, γ-DLen-C2K-DMA,DLen-C2K-DMA, and/or C2-TLinDMA.

Various exemplary embodiments of the cationic lipids of the presentinvention, lipid particles and compositions comprising the same, andtheir use to deliver therapeutic nucleic acids, such as siRNA, tomodulate gene and protein expression and to treat metabolic diseases anddisorders, are described in further detail below.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “Apolipoprotein B” or “ApoB” refers to the main apolipoproteinof chylomicrons and low density lipoproteins (LDL). Mutations in APOBare associated with hypercholesterolemia. ApoB occurs in the plasma in 2main forms: apoB48 and apoB100, which are synthesized in the intestineand liver, respectively, due to an organ-specific stop codon. ApoB48contains 2,152 residues compared to 4,535 residues in apoB100. Cloningand characterization of APOB is described by, e.g., Glickman et al.,PNAS USA 83:5296-5300 (1986); Chen et al., J. Biol. Chem. 261:2918-12921 (1986); and Hospattankar et al., J. Biol. Chem. 261:9102-9104(1986). APOB sequences are set forth in, e.g., Genbank Accession Nos.NM_(—)000384 and BC051278. siRNA sequences that target APOB are setforth herein as well as in U.S. Patent Publication Nos. 20060134189 and20060105976, PCT Publication No. WO 04/091515, Soutschek et al., Nature432:173-178 (2004), and Zimmermann et al., Nature, 441: 111-114 (2006).

The term “interfering RNA” or “RNAi” or “interfering RNA sequence” asused herein includes single-stranded RNA (e.g., mature miRNA, ssRNAioligonucleotides, ssDNAi oligonucleotides), double-stranded RNA (i.e.,duplex RNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, orpre-miRNA), a DNA-RNA hybrid (see, e.g., PCT Publication No. WO2004/078941), or a DNA-DNA hybrid (see, e.g., PCT Publication No. WO2004/104199) that is capable of reducing or inhibiting the expression ofa target gene or sequence (e.g., by mediating the degradation orinhibiting the translation of mRNAs which are complementary to theinterfering RNA sequence) when the interfering RNA is in the same cellas the target gene or sequence. Interfering RNA thus refers to thesingle-stranded RNA that is complementary to a target mRNA sequence orto the double-stranded RNA formed by two complementary strands or by asingle, self-complementary strand. Interfering RNA may have substantialor complete identity to the target gene or sequence, or may comprise aregion of mismatch (i.e., a mismatch motif). The sequence of theinterfering RNA can correspond to the full-length target gene, or asubsequence thereof. Preferably, the interfering RNA molecules arechemically synthesized. The disclosures of each of the above patentdocuments are herein incorporated by reference in their entirety for allpurposes.

Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g.,interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides inlength, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotidesin length, and is preferably about 20-24, 21-22, or 21-23 (duplex)nucleotides in length (e.g., each complementary sequence of thedouble-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25nucleotides in length, preferably about 20-24, 21-22, or 21-23nucleotides in length, and the double-stranded siRNA is about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferablyabout 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes maycomprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 toabout 3 nucleotides and 5′ phosphate termini. Examples of siRNA include,without limitation, a double-stranded polynucleotide molecule assembledfrom two separate stranded molecules, wherein one strand is the sensestrand and the other is the complementary antisense strand; adouble-stranded polynucleotide molecule assembled from a single strandedmolecule, where the sense and antisense regions are linked by a nucleicacid-based or non-nucleic acid-based linker; a double-strandedpolynucleotide molecule with a hairpin secondary structure havingself-complementary sense and antisense regions; and a circularsingle-stranded polynucleotide molecule with two or more loop structuresand a stem having self-complementary sense and antisense regions, wherethe circular polynucleotide can be processed in vivo or in vitro togenerate an active double-stranded siRNA molecule. As used herein, theterm “siRNA” includes RNA-RNA duplexes as well as DNA-RNA hybrids (see,e.g., PCT Publication No. WO 2004/078941).

Preferably, siRNA are chemically synthesized. siRNA can also begenerated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25nucleotides in length) with the E. coli RNase III or Dicer. Theseenzymes process the dsRNA into biologically active siRNA (see, e.g.,Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegariet al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al.,Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res.,31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); andRobertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA areat least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotidesin length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotidesin length, or longer. The dsRNA can encode for an entire gene transcriptor a partial gene transcript. In certain instances, siRNA may be encodedby a plasmid (e.g., transcribed as sequences that automatically foldinto duplexes with hairpin loops).

As used herein, the term “mismatch motif” or “mismatch region” refers toa portion of an interfering RNA (e.g., siRNA) sequence that does nothave 100% complementarity to its target sequence. An interfering RNA mayhave at least one, two, three, four, five, six, or more mismatchregions. The mismatch regions may be contiguous or may be separated by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatchmotifs or regions may comprise a single nucleotide or may comprise two,three, four, five, or more nucleotides.

The phrase “inhibiting expression of a target gene” refers to theability of an interfering RNA (e.g., siRNA) to silence, reduce, orinhibit the expression of a target gene (e.g., APOB, APOC3, PCSK9,DGAT1, and/or DGAT2). To examine the extent of gene silencing, a testsample (e.g., a sample of cells in culture expressing the target gene)or a test mammal (e.g., a mammal such as a human or an animal model suchas a rodent (e.g., mouse) or a non-human primate (e.g., monkey) model)is contacted with an interfering RNA (e.g., siRNA) that silences,reduces, or inhibits expression of the target gene. Expression of thetarget gene in the test sample or test animal is compared to expressionof the target gene in a control sample (e.g., a sample of cells inculture expressing the target gene) or a control mammal (e.g., a mammalsuch as a human or an animal model such as a rodent (e.g., mouse) ornon-human primate (e.g., monkey) model) that is not contacted with oradministered the interfering RNA (e.g., siRNA). The expression of thetarget gene in a control sample or a control mammal may be assigned avalue of 100%. In particular embodiments, silencing, inhibition, orreduction of expression of a target gene is achieved when the level oftarget gene expression in the test sample or the test mammal relative tothe level of target gene expression in the control sample or the controlmammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. In other words, theinterfering RNAs (e.g., siRNAs) of the present invention are capable ofsilencing, reducing, or inhibiting the expression of a target gene(e.g., APOB, APOC3, PCSK9, DGAT1, and/or DGAT2) by at least about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% in a test sample or a test mammal relativeto the level of target gene expression in a control sample or a controlmammal not contacted with or administered the interfering RNA. Suitableassays for determining the level of target gene expression include,without limitation, examination of protein or mRNA levels usingtechniques known to those of skill in the art, such as, e.g., dot blots,Northern blots, in situ hybridization, ELISA, immunoprecipitation,enzyme function, as well as phenotypic assays known to those of skill inthe art.

An “effective amount” or “therapeutically effective amount” of aninterfering RNA is an amount sufficient to produce the desired effect,e.g., an inhibition of expression of a target sequence in comparison tothe normal expression level detected in the absence of an interferingRNA. Inhibition of expression of a target gene or target sequence isachieved when the value obtained with an interfering RNA relative to thecontrol is about 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%,40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays formeasuring expression of a target gene or target sequence include, e.g.,examination of protein or RNA levels using techniques known to those ofskill in the art such as dot blots, northern blots, in situhybridization, ELISA, immunoprecipitation, enzyme function, as well asphenotypic assays known to those of skill in the art.

By “decrease,” “decreasing,” “reduce,” or “reducing” of an immuneresponse by an interfering RNA is intended to mean a detectable decreaseof an immune response to a given interfering RNA (e.g., a modifiedinterfering RNA). The amount of decrease of an immune response by amodified interfering RNA may be determined relative to the level of animmune response in the presence of an unmodified interfering RNA. Adetectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or morelower than the immune response detected in the presence of theunmodified interfering RNA. A decrease in the immune response tointerfering RNA is typically measured by a decrease in cytokineproduction (e.g., IFNγ, IFNα, TNFα, IL-6, IL-8, or IL-12) by a respondercell in vitro or a decrease in cytokine production in the sera of amammalian subject after administration of the interfering RNA.

As used herein, the term “responder cell” refers to a cell, preferably amammalian cell, that produces a detectable immune response whencontacted with an immunostimulatory interfering RNA such as anunmodified siRNA. Exemplary responder cells include, e.g., dendriticcells, macrophages, peripheral blood mononuclear, cells (PBMCs),splenocytes, and the like. Detectable immune responses include, e.g.,production of cytokines or growth factors such as TNF-α, IFN-α, IFN-β,IFN-γ, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13,TGF, and combinations thereof. Detectable immune responses also include,e.g., induction of interferon-induced protein with tetratricopeptiderepeats 1 (IFIT1) mRNA.

“Substantial identity” refers to a sequence that hybridizes to areference sequence under stringent conditions, or to a sequence that hasa specified percent identity over a specified region of a referencesequence.

The phrase “stringent hybridization conditions” refers to conditionsunder which a nucleic acid will hybridize to its target sequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization.

Exemplary stringent hybridization conditions can be as follows: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec.-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y. (1990).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous references, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds.

The terms “substantially identical” or “substantial identity,” in thecontext of two or more nucleic acids, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same (i.e., at least about 60%, preferably atleast about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over aspecified region), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. This definition, when the context indicates, alsorefers analogously to the complement of a sequence. Preferably, thesubstantial identity exists over a region that is at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of a number of contiguous positions selected from the groupconsisting of from about 5 to about 60, usually about 10 to about 45,more usually about 15 to about 30, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.,48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology, Ausubelet al., eds. (1995 supplement)).

Non-limiting examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.,215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). Anotherexample is a global alignment algorithm for determining percent sequenceidentity such as the Needleman-Wunsch algorithm for aligning protein ornucleotide (e.g., RNA) sequences.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNAduplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC,artificial chromosomes), expression cassettes, chimeric sequences,chromosomal DNA, or derivatives and combinations of these groups. RNAmay be in the form of small interfering RNA (siRNA), Dicer-substratedsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA),microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), andcombinations thereof. Nucleic acids include nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,and which have similar binding properties as the reference nucleic acid.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, SNPs, and complementary sequences aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups. “Bases” include purines and pyrimidines, which furtherinclude natural compounds adenine, thymine, guanine, cytosine, uracil,inosine, and natural analogs, and synthetic derivatives of purines andpyrimidines, which include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide(e.g., ApoB).

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids; and (3) “derived lipids” such assteroids.

The term “lipid particle” includes a lipid formulation that can be usedto deliver an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA), to a target site of interest (e.g., cell,tissue, organ, and the like). In preferred embodiments, the lipidparticle of the invention is a nucleic acid-lipid particle, which istypically formed from a cationic lipid, a non-cationic lipid, andoptionally a conjugated lipid that prevents aggregation of the particle.In other preferred embodiments, the active agent or therapeutic agent,such as a nucleic acid, may be encapsulated in the lipid portion of theparticle, thereby protecting it from enzymatic degradation.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a particle made from lipids (e.g., acationic lipid, a non-cationic lipid, and optionally a conjugated lipidthat prevents aggregation of the particle), wherein the nucleic acid(e.g., an interfering RNA) is fully encapsulated within the lipid. Incertain instances, SNALP are extremely useful for systemic applications,as they can exhibit extended circulation lifetimes following intravenous(i.v.) injection, they can accumulate at distal sites (e.g., sitesphysically separated from the administration site), and they can mediatesilencing of target gene expression at these distal sites. The nucleicacid may be complexed with a condensing agent and encapsulated within aSNALP as set forth in PCT Publication No. WO 00/03683, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes.

The lipid particles of the invention (e.g., SNALP) typically have a meandiameter of from about 30 nm to about 150 nm, from about 40 nm to about150 nm, from about 50 nm to about 150 nm; from about 60 nm to about 130nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm,from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In addition, nucleic acids, whenpresent in the lipid particles of the present invention, are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Patent Publication Nos. 20040142025 and 20070042031, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

As used herein, “lipid encapsulated” can refer to a lipid particle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA that targets APOB), with full encapsulation,partial encapsulation, or both. In a preferred embodiment, the nucleicacid is fully encapsulated in the lipid particle (e.g., to form a SNALPor other nucleic acid-lipid particle).

The term “lipid conjugate” refers to a conjugated lipid that inhibitsaggregation of lipid particles. Such lipid conjugates include, but arenot limited to, PEG-lipid conjugates such as, e.g., PEG coupled todialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled todiacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol,PEG coupled to phosphatidylethanolamines, and PEG conjugated toceramides (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids,polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates; see,e.g., U.S. Provisional Application No. 61/294,828, filed Jan. 13, 2010,and U.S. Provisional Application No. 61/295, 140, filed Jan. 14, 2010),polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof.Additional examples of POZ-lipid conjugates are described in PCTPublication No. WO 2010/006282. PEG or POZ can be conjugated directly tothe lipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG or the POZ to a lipid can be usedincluding, e.g., non-ester containing linker moieties andester-containing linker moieties. In certain preferred embodiments,non-ester containing linker moieties, such as amides or carbamates, areused. The disclosures of each of the above patent documents are hereinincorporated by reference in their entirety for all purposes.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long-chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic, or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids, and sphingolipids.

Representative examples of phospholipids include, but are not limitedto, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, are also within the group designated as amphipathiclipids. Additionally, the amphipathic lipids described above can bemixed with other lipids including triglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N-N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The term “fusogenic” refers to the ability of a lipid particle, such asa SNALP, to fuse with the membranes of a cell. The membranes can beeither the plasma membrane or membranes surrounding organelles, e.g.,endosome, nucleus, etc.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

“Distal site,” as used herein, refers to a physically separated site,which is not limited to an adjacent capillary bed, but includes sitesbroadly distributed throughout an organism.

“Serum-stable” in relation to nucleic acid-lipid particles such as SNALPmeans that the particle is not significantly degraded after exposure toa serum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of lipidparticles that leads to a broad biodistribution of an active agent suchas an interfering RNA (e.g., siRNA) within an organism. Some techniquesof administration can lead to the systemic delivery of certain agents,but not others. Systemic delivery means that a useful, preferablytherapeutic, amount of an agent is exposed to most parts of the body. Toobtain broad biodistribution generally requires a blood lifetime suchthat the agent is not rapidly degraded or cleared (such as by first passorgans (liver, lung, etc.) or by rapid, nonspecific cell binding) beforereaching a disease site distal to the site of administration. Systemicdelivery of lipid particles can be by any means known in the artincluding, for example, intravenous, subcutaneous, and intraperitoneal.In a preferred embodiment, systemic delivery of lipid particles is byintravenous delivery.

“Local delivery,” as used herein, refers to delivery of an active agentsuch as an interfering RNA (e.g., siRNA) directly to a target sitewithin an organism. For example, an agent can be locally delivered bydirect injection into a disease site or other target site such as a siteof inflammation or a target organ such as the liver, heart, pancreas,kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

III. Description of the Embodiments

The present invention provides novel, serum-stable lipid particlescomprising one or more therapeutic nucleic acids, methods of making thelipid particles, and methods of delivering and/or administering thelipid particles (e.g., for the treatment of a disease or disorder).

In certain embodiments, the therapeutic nucleic acid comprises aninterfering RNA molecule such as, e.g., an siRNA, Dicer-substrate dsRNA,shRNA, aiRNA, miRNA, or mixtures thereof. In preferred embodiment, theinterfering RNA targets a gene of interest in the liver. Examples oftarget genes of interest that are in the liver include, but are notlimited to, APOB, APOC3, PCSK9, DGAT1, DGAT2, and combinations thereof.

In one aspect, the present invention provides a nucleic acid-lipidparticle (e.g., SNALP) comprising:

(a) an interfering RNA that silences Apolipoprotein B (APOB) expressionand/or the expression of another liver target gene such as APOC3, PCSK9,DGAT1, and/or DGAT2;

(b) a cationic lipid of Formula I having the following structure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently optionally substituted C₁₂-C₂₄ alkyl, optionallysubstituted C₁₂-C₂₄ alkenyl, optionally substituted C₁₂-C₂₄ alkynyl, oroptionally substituted C₁₂-C₂₄ acyl, with the proviso that at least oneof R¹ and R² has at least two sites of unsaturation; R³ and R⁴ areeither the same or different and are independently optionallysubstituted C₁-C₆ alkyl, optionally substituted C₂-C₆ alkenyl, oroptionally substituted C₂-C₆ alkynyl or R³ and R⁴ may join to form anoptionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or2 heteroatoms chosen from nitrogen and oxygen; R⁵ is either absent orhydrogen or C₁-C₆ alkyl to provide a quaternary amine; m, n and p areeither the same or different and are independently either 0, 1 or 2,with the proviso that m, n, and p are not simultaneously 0; q is 0, 1,2, 3, or 4; Y and Z are either the same or different and areindependently O, S, or NH; and

(c) a non-cationic lipid.

In another aspect, the present invention provides a nucleic acid-lipidparticle (e.g., SNALP) comprising:

(a) an interfering RNA that silences Apolipoprotein B (APOB) expressionand/or the expression of another liver target gene such as APOC3, PCSK9,DGAT1, and/or DGAT2;

(b) a cationic lipid of Formula II having the following structure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently optionally substituted C₁₂-C₂₄ alkyl, optionallysubstituted C₁₂-C₂₄ alkenyl, optionally substituted C₁₂-C₂₄ alkynyl, oroptionally substituted C₁₂-C₂₄ acyl; R³ and R⁴ are either the same ordifferent and are independently optionally substituted C₁-C₆ alkyl,optionally substituted C₂-C₆ alkenyl, or optionally substituted C₂-C₆alkynyl or R³ and R⁴ may join to form an optionally substitutedheterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosenfrom nitrogen and oxygen; R⁵ is either absent or is hydrogen or C₁-C₆alkyl to provide a quaternary amine; m, n, and p are either the same ordifferent and are independently either 0, 1 or 2, with the proviso thatm, n, and p are not simultaneously 0; Y and Z are either the same ordifferent and are independently O, S, or NH; and

(c) a non-cationic lipid.

In some embodiments, cationic lipids falling within the scope ofFormulas I and/or II that are useful in the nucleic acid-lipid particlesof the present invention (e.g., SNALP) include, but are not limited to,the following: 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-K-C2-DMA; “XTC2” or “C2K”),2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA;“C3K”), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane(DLin-K-C4-DMA; “C4K”),2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA),2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DO-K-DMA),2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DS-K-DMA),2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane (DLin-K-MA),2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride(DLin-K-TMA.Cl), 2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane (DLin-K²-DMA),2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane(D-Lin-K-N-methylpiperzine), analogs thereof, salts thereof, andmixtures thereof. In preferred embodiments, the cationic lipid comprisesDLin-K-C2-DMA (“C2K”).

In particular embodiments, the interfering RNA (e.g., siRNA) thattargets APOB and/or other target genes such as APOC3, PCSK9, DGAT1,and/or DGAT2 comprises a sense strand and a complementary antisensestrand, and the interfering RNA comprises a double-stranded region ofabout 15 to about 60 nucleotides in length (e.g., about 15-60, 15-30,15-25, 19-30, 19-25, 20-60, 20-55, 20-50, 20-45, 20-40, 20-35, 20-30,20-25, 21-30, 21-29, 22-30, 22-29, 22-28, 23-30, 23-28, 24-30, 24-28,25-60, 25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides inlength, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, or 35 nucleotides in length). In one embodiment,the interfering RNA is chemically synthesized. The interfering RNAmolecules of the invention are capable of silencing the expression of atarget sequence such as APOB in vitro and/or in vivo.

In certain embodiments, the interfering RNA (e.g., siRNA) of the presentinvention may comprise at least one, two, three, four, five, six, seven,eight, nine, ten, or more modified nucleotides such as 2′OMenucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region of the interfering RNA. Preferably, uridineand/or guanosine nucleotides in the interfering RNA are modified with2′OMe nucleotides. In certain instances, the interfering RNA contains2′OMe nucleotides in both the sense and antisense strands and comprisesat least one 2′OMe-uridine nucleotide and at least one 2′OMe-guanosinenucleotide in the double-stranded region. In some embodiments, the senseand/or antisense strand of the interfering RNA may further comprisemodified (e.g., 2′OMe-modified) adenosine and/or modified (e.g.,2′OMe-modified) cytosine nucleotides, e.g., in the double-strandedregion of the interfering RNA.

In some embodiments, the sense and/or antisense strand sequences maycomprise at least one, two, three, four, five, six, seven, eight, nine,ten, or more modified nucleotides such as 2′OMe nucleotides. In certainembodiments, the sense and/or antisense strand sequences may eachindependently comprise or consist of a modified (e.g., 2′OMe) and/orunmodified 3′ overhang of 1, 2, 3, of 4 nucleotides, or one or both endsof the double-stranded molecule may be blunt-ended.

One of skill in the art will understand that unmodified sense and/orantisense strand sequences can be modified in accordance with theselective modification patterns described herein (e.g., at selectiveuridine and/or guanosine nucleotides, and optionally at adenosine and/orcytosine nucleotides, within the RNA duplex), and screened for RNAiactivity as well as immune stimulation, such that the degree of chemicalmodifications introduced into the interfering RNA molecule strikes abalance between reduction or abrogation of the immunostimulatoryproperties of the interfering RNA and retention of RNAi activity.

In particular embodiments, the interfering RNA (e.g., siRNA) moleculesof the present invention comprise a 3′ overhang of 1, 2, 3, or 4nucleotides in one or both strands. In certain instances, theinterfering RNA may contain at least one blunt end. In particularembodiments, the 3′ overhangs in one or both strands of the interferingRNA may each independently comprise 1, 2, 3, or 4 modified and/orunmodified deoxythymidine (“t” or “dT”) nucleotides, 1, 2, 3, or 4modified (e.g., 2′OMe) and/or unmodified uridine (“U”) ribonucleotides,or 1, 2, 3, or 4 modified (e.g., 2′OMe) and/or unmodifiedribonucleotides or deoxyribonucleotides having complementarity to thetarget sequence or the complementary strand thereof.

In another embodiment, the present invention provides a compositioncomprising a cocktail (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, or more) of unmodified and/ormodified interfering RNA (e.g., siRNA) sequences that target APOB,APOC3, PCSK9, DGAT1, and/or DGAT2 expression. The cocktail ofinterfering RNA (e.g., siRNA) may comprise sequences which are directedto the same region or domain (e.g., a “hot spot”) and/or to differentregions or domains of one or more target genes. In particularembodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more (e.g., all) of these sequences arechemically modified (e.g., 2′OMe-modified) as described herein.

In certain embodiments, the sense strand comprises or consists of asequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the targetsequence or a portion thereof. In certain other embodiments, the sensestrand comprises or consists of at least about 15 contiguous nucleotides(e.g., at least about 15, 16, 17, 18, or 19 contiguous nucleotides) of asequence that is identical to the target sequence or a portion thereof.In preferred embodiments, the interfering RNA (e.g., siRNA) comprisingsuch a sense strand sequence is capable of mediating target-specificRNAi.

In some embodiments, the antisense strand comprises or consists of asequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to thetarget sequence or a portion thereof. In other embodiments, theantisense strand comprises or consists of at least about 15 contiguousnucleotides (e.g., at least about 15, 16, 17, 18, or 19 contiguousnucleotides) of a sequence that is complementary to the target sequenceor a portion thereof. In further embodiments, the antisense strandcomprises or consists of a sequence that specifically hybridizes to thetarget sequence or a portion thereof. In preferred embodiments, theinterfering RNA (e.g., siRNA) comprising such an antisense strandsequence is capable of mediating target-specific RNAi.

In one preferred embodiment, the APOB siRNA comprises an antisensestrand comprising the following sequence: 5′-UAUUCAGUGUGAUGACACU-3′. Inanother preferred embodiment, the APOB siRNA further comprises a sensestrand comprising the following sequence: 5′-AGUGUCAUCACACUGAAUA-3′. Insome embodiments, the APOB siRNA comprises at least one 2′OMenucleotide, e.g., at least one 2′OMe-guanosine and/or 2′OMe-uridinenucleotide. In certain instances, the APOB siRNA comprises an antisensestrand comprising at least one, at least two, at least three, at leastfour, at least five, at least six, at least seven, or more 2′OMenucleotides, e.g., 2′OMe-guanosine and/or 2′OMe-uridine nucleotides. Incertain other instances, the APOB siRNA comprises a sense strandcomprising at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, or more 2′OMe nucleotides,e.g., 2′OMe-guanosine and/or 2′OMe-uridine nucleotides.

In particular embodiments, from about 20%-40%, 25%-40%, 30%-40%,20%-35%, 25%-35%, 20%-30%, 25%-30%, 26%-34%, 27%-33%, 28%-32%, or about25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,39%, or 40% of the nucleotides in the double-stranded region of thesiRNA comprise modified nucleotides such as, e.g., 2′OMe nucleotides(e.g., 2′OMe-guanosine and/or 2′OMe-uridine nucleotides).

In some embodiments, the APOB siRNA of the invention comprises a 3′overhang in one or both strands of the siRNA. In one particularembodiment, the antisense strand comprises a 5′-UC-3′ overhang and thesense strand comprises a 5′-CC-3′ overhang. In certain instances, the 3′overhangs on one or both strands of the siRNA comprise at least one2′OMe nucleotide, e.g., at least one 2′OMe-guanosine and/or2′OMe-uridine nucleotide. In other embodiments, the 3′ overhangs on oneor both strands of the siRNA molecule comprise 1-4 deoxythymidine (dT)nucleotides, 1-4 modified and/or unmodified uridine (U) ribonucleotides,or 1-2 additional ribonucleotides having complementarity to the targetsequence or the complementary strand thereof.

In a first embodiment, the APOB siRNA comprises the following sensestrand sequence: 5′-AGUGUCAUCACACUGAAUACC-3′ (“S-1”), wherein the boldedand underlined nucleotides are 2′OMe nucleotides. In a secondembodiment, the APOB siRNA comprises the following sense strandsequence: 5′-AGUGUCAUCACACUGAAUACC-3′(“S-2”), wherein the bolded andunderlined nucleotides are 2′OMe nucleotides. In a third embodiment, theAPOB siRNA comprises the following sense strand sequence:5′-AGUGUCAUCACACUGAAUACC-3′ (“S-3”), wherein the bolded and underlinednucleotides are 2′OMe nucleotides. In a fourth embodiment, the APOBsiRNA comprises the following sense strand sequence:5′-AGUGUCAUCACACUGAAUACC-3′ (“S-4”), wherein the bolded and underlinednucleotides are 2′OMe nucleotides. In a fifth embodiment, the APOB siRNAcomprises the following sense strand sequence:5′-AGUGUCAUCACACUGAAUACC-3′ (“S-5”), wherein the bolded and underlinednucleotides are 2′OMe nucleotides. In a sixth embodiment, the APOB siRNAcomprises the following sense strand sequence:5′-AGUGUCAUCACACUGAAUACC-3′ (“S-6”), wherein the bolded and underlinednucleotides are 2′OMe nucleotides.

In a first embodiment, the APOB siRNA comprises the following antisensestrand sequence: 5′-UAUUCAGUGUGAUGACACUUG-3′ (“AS-1”), wherein thebolded and underlined nucleotides are 2′OMe nucleotides. In a secondembodiment, the APOB siRNA comprises the following antisense strandsequence: 5′-UAUUCAGUGUGAUGACACUUG-3′ (“AS-2”), wherein the bolded andunderlined nucleotides are 2′OMe nucleotides. In a third embodiment, theAPOB siRNA comprises the following antisense strand sequence:5′-UAUUCAGUGUGAUGACACUUG-3′ (“AS-3”), wherein the bolded and underlinednucleotides are 2′OMe nucleotides. In a fourth embodiment, the APOBsiRNA comprises the following antisense strand sequence:5′-UAUUCAGUGUGAUGACACUUG-3′ (“AS-4”), wherein the bolded and underlinednucleotides are 2′OMe nucleotides. In a fifth embodiment, the APOB siRNAcomprises the following antisense strand sequence:5′-UAUUCAGUGUGAUGACACUUG-3′ (“AS-5”), wherein the bolded and underlinednucleotides are 2′OMe nucleotides. In a sixth embodiment, the APOB siRNAcomprises the following antisense strand sequence:5′-UAUUCAGUGUGAUGACACUUG-3′ (“AS-6”), wherein the bolded and underlinednucleotides are 2′OMe nucleotides.

In one preferred embodiment, the APOB siRNA comprises: an antisensestrand comprising the sequence 5′-UAUUCAGUGUGAUGACACU-3′ and at leastone, two, three, four, five, six, or more 2′OMe nucleotides, e.g., atleast one, two, three, four, five, six, or more 2′OMe-guanosine and/or2′OMe-uridine nucleotides; and a sense strand comprising the sequence5′-AGUGUCAUCACACUGAAUA-3′ and at least one, two, three, four, five, six,or more 2′OMe nucleotides, e.g., at least one, two, three, four, five,six, or more 2′OMe-guanosine and/or 2′OMe-uridine nucleotides. Inanother preferred embodiment, the APOB siRNA of the invention comprises:a sense strand comprising nucleotides 1-19 of S-1, S-2, S-3, S-4, S-5,or S-6; and an antisense strand comprising nucleotides 1-19 of AS-1,AS-2, AS-3, AS-4, AS-5, or AS-6. In a particularly preferred embodiment,the APOB siRNA consists of: a sense strand selected from S-1, S-2, S-3,S-4, S-5, and S-6; and an antisense strand selected from AS-1, AS-2,AS-3, AS-4, AS-5, and AS-6.

In one particular embodiment, the APOB siRNA consists of the followingsense and antisense strand sequences:

5′-AGU G UCA U CACAC U GAAUACC-3′ 3′-GU U CACAGUAGU G U G AC U UAU-5′,(“S-1+AS-1”, “1/1”, or “ApoB-8”), wherein the bolded and underlinednucleotides are 2′OMe nucleotides.

In another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-AG U G U CA U CACAC U GAA U ACC-3′ 3′-GU U CACAG U AG U G U GAC UUAU-5′,(“S-2+AS-2” or “2/2”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In yet another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-AG U G U CA U CACAC U GAA U ACC-3′ 3′-GU U CACAGUA G U G U G AC UUAU-5′,(“S-2+AS-3” or “2/3”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In still yet another particular embodiment, the APOB siRNA consists ofthe following sense and antisense strand sequences:

5′-AG U G U CA U CACAC UG AA U ACC-3′ 3′-GU U CACAG U AG U G U GAC UUAU-5′,(“S-3+AS-2” or “3/2”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-AG U G U CA U CACAC UG AA U ACC-3′ 3′-GU U CACAGUA G U G U G AC UUAU-5′,(“S-3+AS-3” or “3/3”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In yet another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-A G U G UCA U CACACU G AA U ACC-3′ 3′-GU U CACAG U AG U G U GAC UUAU-5′,(“S-4+AS-2” or “4/2”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In still yet another particular embodiment, the APOB siRNA consists ofthe following sense and antisense strand sequences:

5′-A G U G UCA U CACACU G AA U ACC-3′ 3′-GU U CACAGUA G U G U G AC UUAU-5′,(“S-4+AS-3” or “4/3”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-A GUGU CA U CACAC U GAA U ACC-3′ 3′-GU U CACAG U AG U G U GAC UUAU-5′,(“S-5+AS-2” or “5/2”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In yet another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-A GUGU CA U CACAC U GAA U ACC-3′ 3′-GU U CACAGUA G U G U G AC UUAU-5′,(“S-5+AS-3” or “5/3”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In still yet another particular embodiment, the APOB siRNA consists ofthe following sense and antisense strand sequences:

5′-A GU G U CA U CACAC UG AA U ACC-3′ 3′-GU U CACAG U AG U G U GAC UUAU-5′,(“S-6+AS-2” or “6/2”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-A GU G U CA U CACAC UG AA U ACC-3′ 3′-GU U CACAGUA G U G U G AC UUAU-5′,(“S-6+AS-3” or “6/3”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In yet another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-AG U G U CA U CACAC U GAA U ACC-3′ 3′- GUU CACAGUAGU G U G AC UUAU-5′,(“S-2+AS-4” or “2/4”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In still yet another particular embodiment, the APOB siRNA consists ofthe following sense and antisense strand sequences:

5′-AG U G U CA U CACAC U GAA U ACC-3′ 3′- GUU CACAG U AG U G U GAC UUAU-5′,(“S-2+AS-5” or “2/5”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-AG U G U CA U CACAC U GAA U ACC-3′ 3′- GUU CACAGUA G U G U G AC UUAU-5′,(“S-2+AS-6” or “2/6”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In yet another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-AG U G U CA U CACAC UG AA U ACC-3′ 3′- GUU CACAGUAGU G U G AC UUAU-5′,(“S-3+AS-4”, or “3/4”), wherein the bolded and underlined nucleotidesare 2′OMe nucleotides.

In still yet another particular embodiment, the APOB siRNA consists ofthe following sense and antisense strand sequences:

5′-AG U G U CA U CACAC UG AA U ACC-3′ 3′- GUU CACAG U AG U G U GAC UUAU-5′,(“S-3+AS-5”, “3/5”, or “ApoB-10”), wherein the bolded and underlinednucleotides are 2′OMe nucleotides.

In another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-AG U G U CA U CACAC UG AA U ACC-3′ 3′- GUU CACAGUA G U G U G AC UUAU-5′,(“S-3+AS-6” or “3/6”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In yet another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-A G U G UCA U CACACU G AA U ACC-3′ 3′- GUU CACAGUAGU G U G AC UUAU-5′,(“S-4+AS-4” or “4/4”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In still yet another particular embodiment, the APOB siRNA consists ofthe following sense and antisense strand sequences:

5′-A G U G UCA U CACACU G AA U ACC-3′ 3′- GUU CACAG U AG U G U GAC UUAU-5′,(“S-4+AS-5” or “4/5”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-A G U G UCA U CACACU G AA U ACC-3′ 3′- GUU CACAGUA G U G U G AC UUAU-5′,(“S-4+AS-6” or “4/6”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In yet another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-A GUGU CA U CACAC U GAA U ACC-3′ 3′- GUU CACAGUAGU G U G AC U UAU-5′,(“S-5+AS-4” or “5/4”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In still yet another particular embodiment, the APOB siRNA consists ofthe following sense and antisense strand sequences:

5′-A GUGU CA U CACAC U GAA U ACC-3′ 3′- GUU CACAG U AG U G U GAC UUAU-5′,(“5-5+AS-5” or “5/5”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-A GUGU CA U CACAC U GAA U ACC-3′ 3′- GUU CACAGUA G U G U G AC UUAU-5′,(“S-5+AS-6” or “5/6”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In yet another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-A GU G U CA U CACAC UG AA U ACC-3′ 3′- GUU CACAGUAGU G U G AC UUAU-5′,(“S-6+AS-4” or “6/4”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In still yet another particular embodiment, the APOB siRNA consists ofthe following sense and antisense strand sequences:

5′-A GU G U CA U CACAC UG AA U ACC-3′ 3′- GUU CACAG U AG U G U GAC UUAU-5′,(“S-6+AS-5” or “6/5”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In another particular embodiment, the APOB siRNA consists of thefollowing sense and antisense strand sequences:

5′-A GU G U CA U CACAC UG AA U ACC-3′ 3′- GUU CACAGUA G U G U G AC UUAU-5′,(“S-6+AS-6” or “6/6”), wherein the bolded and underlined nucleotides are2′OMe nucleotides.

In a further embodiment, the APOB siRNA consists of the following senseand antisense strand sequences:

5′-GUCAUCACACUGAAUACCAAU-3′ 3′-CACAGUAGUGUGACUUAUGGUUA-5′.It will be readily apparent to those of skill in the art that theforegoing APOB siRNA can also be chemically modified, if desired, toreduce its immunostimulatory properties, while maintaining its silencingactivities.

The nucleic acid-lipid particles (e.g., SNALP) typically comprise one ormore (e.g., a cocktail) of the interfering RNAs described herein, acationic lipid, and a non-cationic lipid. In certain instances, thenucleic acid-lipid particles (e.g., SNALP) further comprise a conjugatedlipid that inhibits aggregation of particles. Preferably, the nucleicacid-lipid particles (e.g., SNALP) comprise one or more (e.g., acocktail) of the interfering RNAs described herein, a cationic lipid, anon-cationic lipid, and a conjugated lipid that inhibits aggregation ofparticles. In particular embodiments, the nucleic acid-lipid particles(e.g., SNALP) of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, or moreunmodified and/or modified interfering RNAs that silence 1, 2, 3, 4, 5,6, 7, 8, or more different genes associated with liver diseases ordisorders, a cationic lipid, a non-cationic lipid, and a conjugatedlipid that inhibits aggregation of particles.

In some embodiments, the interfering RNAs (e.g., siRNAs) are fullyencapsulated in the nucleic acid-lipid particle (e.g., SNALP). Withrespect to formulations comprising an interfering RNA cocktail, thedifferent types of interfering RNA species present in the cocktail(e.g., interfering RNA compounds with different sequences) may beco-encapsulated in the same particle, or each type of interfering RNAspecies present in the cocktail may be encapsulated in a separateparticle. The interfering RNA cocktail may be formulated in theparticles described herein using a mixture of two or more individualinterfering RNAs (each having a unique sequence) at identical, similar,or different concentrations or molar ratios. In one embodiment, acocktail of interfering RNAs (corresponding to a plurality ofinterfering RNAs with different sequences) is formulated usingidentical, similar, or different concentrations or molar ratios of eachinterfering RNA species, and the different types of interfering RNAs areco-encapsulated in the same particle. In another embodiment, each typeof interfering RNA species present in the cocktail is encapsulated indifferent particles at identical, similar, or different interfering RNAconcentrations or molar ratios, and the particles thus formed (eachcontaining a different interfering RNA payload) are administeredseparately (e.g., at different times in accordance with a therapeuticregimen), or are combined and administered together as a single unitdose (e.g., with a pharmaceutically acceptable carrier). The particlesdescribed herein are serum-stable, are resistant to nucleasedegradation, and are substantially non-toxic to mammals such as humans.

The cationic lipid in the nucleic acid-lipid particles of the invention(e.g., SNALP) may comprise, e.g., one or more cationic lipids of FormulaI and II described herein and/or any other cationic lipid species. Inone particular embodiment, the cationic lipid comprises2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-K-C2-DMA or“C2K”).

In addition to the cationic lipids of Formula I and II, the cationiclipids in the nucleic acid-lipid particles of the invention (e.g.,SNALP) may comprise, e.g., one or more cationic lipids of FormulaIII-XIV (or salts thereof) described herein, either alone or incombination with other known cationic lipids. In one particularembodiment, the cationic lipid comprises2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-K-C2-DMA),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), or a mixturethereof.

The non-cationic lipid in the nucleic acid-lipid particles of thepresent invention (e.g., SNALP) may comprise, e.g., one or more anioniclipids and/or neutral lipids. In some embodiments, the non-cationiclipid comprises one of the following neutral lipid components: (1) amixture of a phospholipid and cholesterol or a derivative thereof; (2)cholesterol or a derivative thereof; or (3) a phospholipid. In certainpreferred embodiments, the phospholipid comprisesdipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), or a mixture thereof. In a particularly preferred embodiment,the non-cationic lipid is a mixture of DPPC and cholesterol.

The lipid conjugate in the nucleic acid-lipid particles of the invention(e.g., SNALP) inhibits aggregation of particles and may comprise, e.g.,one or more of the lipid conjugates described herein. In one particularembodiment, the lipid conjugate comprises a PEG-lipid conjugate.Examples of PEG-lipid conjugates include, but are not limited to,PEG-DAG conjugates, PEG-DAA conjugates, and mixtures thereof. In certainembodiments, the PEG-DAA conjugate in the lipid particle may comprise aPEG-didecyloxypropyl (C₁₀) conjugate, a PEG-dilauryloxypropyl (C₁₂)conjugate, a PEG-dimyristyloxypropyl (C₁₄) conjugate, aPEG-dipalmityloxypropyl (C₁₆) conjugate, a PEG-distearyloxypropyl (C₁₈)conjugate, or mixtures thereof. In another embodiment, the lipidconjugate comprises a POZ-lipid conjugate such as a POZ-DAA conjugate.

In exemplary aspects of these embodiments, the cationic lipid isDLin-K-C2-DMA (“C2K”), the non-cationic lipid is a mixture of aphospholipid (e.g., DPPC) and cholesterol, and the PEG-lipid conjugateis a PEG-DAA conjugate such as a PEG2000-DMA and/or a PEG750-DMAconjugate. In a particularly preferred embodiment, the APOB siRNA isAPOB siRNA 3/5 (“ApoB-10”), the cationic lipid is. DLin-K-C2-DMA(“C2K”), the non-cationic lipid is a mixture of a phospholipid (e.g.,DPPC) and cholesterol, and the PEG-lipid conjugate is a PEG-DMAconjugate such as PEG2000-C-DMA.

In certain embodiments, the present invention provides nucleicacid-lipid particles (e.g., SNALP) comprising: (a) one or moreinterfering RNA molecules that target APOB expression and/or theexpression of other liver target genes such as APOC3, PCSK9, DGAT1,DGAT2, or combinations thereof; (b) one or more cationic lipids ofFormula I-XIV or salts thereof comprising from about 50 mol % to about85 mol % of the total lipid present in the particle; (c) one or morenon-cationic lipids comprising from about 13 mol % to about 49.5 mol %of the total lipid present in the particle; and (d) one or moreconjugated lipids that inhibit aggregation of particles comprising fromabout 0.5 mol % to about 2 mol % of the total lipid present in theparticle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) an interfering RNA that targets APOB expression and/orthe expression of another liver target gene such as APOC3, PCSK9, DGAT1,or DGAT2; (b) a cationic lipid of Formula I-XIV or a salt thereofcomprising from about 52 mol % to about 62 mol % of the total lipidpresent in the particle; (c) a mixture of a phospholipid and cholesterolor a derivative thereof comprising from about 36 mol % to about 47 mol %of the total lipid present in the particle; and (d) a PEG-lipidconjugate comprising from about 1 mol % to about 2 mol % of the totallipid present in the particle. This embodiment of nucleic acid-lipidparticle is generally referred to herein as the “1:57” formulation. Inone particular embodiment, the 1:57 formulation is a four-componentsystem comprising about 1.4 mol % PEG-lipid conjugate (e.g.,PEG2000-C-DMA), about 57.1 mol % cationic lipid of Formula I-XIV or asalt thereof, about 7.1 mol % DPPC (or DSPC), and about 34.3 mol %cholesterol (or derivative thereof). In certain embodiments of the 1:57formulation, the cationic lipid is DLin-K-C2-DMA.

In another aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) an interfering RNA that targets APOB expression and/orthe expression of another liver target gene such as APOC3, PCSK9, DGAT1,or DGAT2; (b) a cationic lipid of Formula I-XIV or a salt thereofcomprising from about 56.5 mol % to about 66.5 mol % of the total lipidpresent in the particle; (c) cholesterol or a derivative thereofcomprising from about 31.5 mol % to about 42.5 mol % of the total lipidpresent in the particle; and (d) a PEG-lipid conjugate comprising fromabout 1 mol % to about 2 mol % of the total lipid present in theparticle. This embodiment of nucleic acid-lipid particle is generallyreferred to herein as the “1:62” formulation. In one particularembodiment, the 1:62 formulation is a three-component system which isphospholipid-free and comprises about 1.5 mol % PEG-lipid conjugate(e.g., PEG2000-C-DMA), about 61.5 mol % cationic lipid of Formula I-XIVor a salt thereof, and about 36.9 mol % cholesterol (or derivativethereof). In certain embodiments of the 1:62 formulation, the cationiclipid is DLin-K-C2-DMA.

Additional embodiments related to the 1:57 and 1:62 formulations aredescribed in PCT Publication No. WO 09/127,060 and U.S. application Ser.No. 12/794,701, filed Jun. 4, 2010, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

In other embodiments, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) comprising: (a) one or more interfering RNAmolecules that target APOB expression and/or the expression of otherliver target genes such as APOC3, PCSK9, DGAT1, DGAT2, or combinationsthereof; (b) one or more cationic lipids of Formula I-XIV or saltsthereof comprising from about 2 mol % to about 50 mol % of the totallipid present in the particle; (c) one or more non-cationic lipidscomprising from about 5 mol % to about 90 mol % of the total lipidpresent in the particle; and (d) one or more conjugated lipids thatinhibit aggregation of particles comprising from about 0.5 mol % toabout 20 mol % of the total lipid present in the particle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) an interfering RNA that targets APOB expression and/orthe expression of another liver target gene such as APOC3, PCSK9, DGAT1,or DGAT2; (b) a cationic lipid of Formula I-XIV or a salt thereofcomprising from about 30 mol % to about 50 mol % of the total lipidpresent in the particle; (c) a mixture of a phospholipid and cholesterolor a derivative thereof comprising from about 47 mol % to about 69 mol %of the total lipid present in the particle; and (d) a PEG-lipidconjugate comprising from about 1 mol % to about 3 mol % of the totallipid present in the particle. This embodiment of nucleic acid-lipidparticle is generally referred to herein as the “2:40” formulation. Inone particular embodiment, the 2:40 formulation is a four-componentsystem which comprises about 2 mol % PEG-lipid conjugate (e.g.,PEG2000-C-DMA), about 40 mol % cationic lipid of Formula I-XIV or a saltthereof, about 10 mol % DPPC (or DSPC), and about 48 mol % cholesterol(or derivative thereof). In certain embodiments of the 2:40 formulation,the cationic lipid is DLin-K-C2-DMA.

In further embodiments, the present invention provides nucleicacid-lipid particles (e.g., SNALP) comprising: (a) one or moreinterfering RNA molecules that target APOB expression and/or theexpression of other liver target genes such as APOC3, PCSK9, DGAT1,DGAT2, or combinations thereof; (b) one or more cationic lipids ofFormula I-XIV or salts thereof comprising from about 50 mol % to about65 mol % of the total lipid present in the particle; (c) one or morenon-cationic lipids comprising from about 25 mol % to about 45 mol % ofthe total lipid present in the particle; and (d) one or more conjugatedlipids that inhibit aggregation of particles comprising from about 5 mol% to about 10 mol % of the total lipid present in the particle.

In one aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) an interfering RNA that targets APOB expression and/orthe expression of another liver target gene such as APOC3, PCSK9, DGAT1,or DGAT2; (b) a cationic lipid of Formula I-XIV or a salt thereofcomprising from about 50 mol % to about 60 mol % of the total lipidpresent in the particle; (c) a mixture of a phospholipid and cholesterolor a derivative thereof comprising from about 35 mol % to about 45 mol %of the total lipid present in the particle; and (d) a PEG-lipidconjugate comprising from about 5 mol % to about 10 mol % of the totallipid present in the particle. This embodiment of nucleic acid-lipidparticle is generally referred to herein as the “7:54” formulation. Incertain instances, the non-cationic lipid mixture in the 7:54formulation comprises: (i) a phospholipid of from about 5 mol % to about10 mol % of the total lipid present in the particle; and (ii)cholesterol or a derivative thereof of from about 25 mol % to about 35mol % of the total lipid present in the particle. In one particularembodiment, the 7:54 formulation is a four-component system whichcomprises about 7 mol % PEG-lipid conjugate (e.g., PEG750-C-DMA), about54 mol % cationic lipid of Formula I-XIV or a salt thereof, about 7 mol% DPPC (or DSPC), and about 32 mol % cholesterol (or derivativethereof). In certain embodiments of the 7:54 formulation, the cationiclipid is DLin-K-C2-DMA.

In another aspect of this embodiment, the nucleic acid-lipid particlecomprises: (a) an interfering RNA that targets APOB expression and/orthe expression of another liver target gene such as APOC3, PCSK9, DGAT1,or DGAT2; (b) a cationic lipid of Formula I-XIV or a salt thereofcomprising from about 55 mol % to about 65 mol % of the total lipidpresent in the particle; (c) cholesterol or a derivative thereofcomprising from about 30 mol % to about 40 mol % of the total lipidpresent in the particle; and (d) a PEG-lipid conjugate comprising fromabout 5 mol % to about 10 mol % of the total lipid present in theparticle. This embodiment of nucleic acid-lipid particle is generallyreferred to herein as the “7:58” formulation. In one particularembodiment, the 7:58 formulation is a three-component system which isphospholipid-free and comprises about 7 mol % PEG-lipid conjugate (e.g.,PEG750-C-DMA), about 58 mol % cationic lipid of Formula I-XIV or a saltthereof, and about 35 mol % cholesterol (or derivative thereof). Incertain embodiments of the 7:58 formulation, the cationic lipid isDLin-K-C2-DMA.

Additional embodiments related to the 7:54 and 7:58 formulations aredescribed in U.S. application Ser. No. ______, entitled “Novel LipidFormulations for Delivery of Therapeutic Agents to Solid Tumors,” filedJun. 30, 2010, bearing Attorney Docket No. 020801-009610US, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

The present invention also provides pharmaceutical compositionscomprising a nucleic acid-lipid particle such as a SNALP and apharmaceutically acceptable carrier.

The nucleic acid-lipid particles of the invention are useful for thetherapeutic delivery of interfering RNA (e.g., siRNA) molecules thatsilence the expression of one or more genes associated with liverdiseases or disorders (e.g., APOB, APOC3, PCSK9, DGAT1, and/or DGAT2).In some embodiments, a cocktail of siRNAs that target one or more genesexpressed in the liver is formulated into the same or different nucleicacid-lipid particles, and the particles are administered to a mammal(e.g., a human) requiring such treatment. In certain instances, atherapeutically effective amount of the nucleic acid-lipid particles canbe administered to the mammal, e.g., for treating, preventing, reducingthe risk of developing, or delaying the onset of a lipid disorder suchas dyslipidemia (e.g., elevated triglyceride and/or cholesterol levels)or atherosclerosis.

Non-limiting examples of lipid disorders suitable for prevention and/ortreatment with the nucleic acid-lipid particles of the invention (e.g.,SNALP) include dyslipidemia (e.g., hyperlipidemias such as elevatedtriglyceride levels (hypertriglyceridemia) and/or elevated cholesterollevels (hypercholesterolemia)), atherosclerosis, low HDL-cholesterol,high LDL-cholesterol, coronary heart disease, coronary artery disease,atherosclerotic cardiovascular disease (CVD), fatty liver disease(hepatic steatosis), abnormal lipid metabolism, abnormal cholesterolmetabolism, pancreatitis (e.g., acute pancreatitis associated withsevere hypertriglyceridemia), diabetes (including Type 2 diabetes),obesity, cardiovascular disease, and other disorders relating toabnormal metabolism.

As described in the Examples below, it has surprisingly been found thatthe SNALP formulations of the present invention containing at least onecationic lipid of Formulas I-XIV, either alone or in combination withother cationic lipids, show increased potency when targeting a gene ofinterest in the liver, such as APOB, when compared to other SNALPformulations. Thus, the present invention provides methods for treatinga disease or disorder associated with overexpression of APOB in a mammal(e.g., human) in need thereof, the method comprising administering tothe mammal a therapeutically effective amount of a lipid particle (e.g.,SNALP) comprising one or more interfering RNAs that silence APOBexpression. Diseases and disorders associated with overexpression ofAPOB are described herein and include, but are not limited to,atherosclerosis, angina pectoris, high blood pressure, diabetes, andhypothyroidism. In certain instances, the mammal (e.g., human) has adisease or disorder involving hypercholesterolemia and serum cholesterollevels are lowered when expression of APOB is silenced by theinterfering RNA.

In some embodiments, the interfering RNA (e.g., siRNA) moleculesdescribed herein are used in methods for silencing APOB, APOC3, PCSK9,DGAT1, and/or DGAT2 gene expression, e.g., in a cell such as a livercell. In particular, it is an object of the invention to provide methodsfor treating, preventing, reducing the risk of developing, or delayingthe onset of a lipid disorder in a mammal by downregulating or silencingthe transcription and/or translation of the APOB, APOC3, PCSK9, DGAT1,and/or DGAT2 gene. In certain embodiments, the present inventionprovides a method for introducing one or more interfering RNA (e.g.,siRNA) molecules described herein into a cell by contacting the cellwith a nucleic acid-lipid particle described herein (e.g., a SNALPformulation). In one particular embodiment, the cell is a liver cellsuch as, e.g., a hepatocyte present in the liver tissue of a mammal(e.g., a human). In another embodiment, the present invention provides amethod for the in vivo delivery of one or more interfering RNA (e.g.,siRNA) molecules described herein to a liver cell (e.g., hepatocyte) byadministering to a mammal (e.g., human) a nucleic acid-lipid particledescribed herein (e.g., a SNALP formulation).

In some embodiments, the nucleic acid-lipid particles described herein(e.g., SNALP) are administered by one of the following routes ofadministration: oral, intranasal, intravenous, intraperitoneal,intramuscular, intra-articular, intralesional, intratracheal,subcutaneous, and intradermal. In particular embodiments, the nucleicacid-lipid particles are administered systemically, e.g., via enteral orparenteral routes of administration.

In particular embodiments, the nucleic acid-lipid particles of theinvention (e.g., SNALP) can preferentially deliver a payload such as aninterfering RNA (e.g., siRNA) to the liver as compared to other tissues,e.g., for the treatment of a liver disease or disorder such asdyslipidemia or atherosclerosis.

In certain aspects, the present invention provides methods for silencingAPOB, APOC3, PCSK9, DGAT1, and/or DGAT2 gene expression in a mammal(e.g., human) in need thereof, the method comprising administering tothe mammal a therapeutically effective amount of a nucleic acid-lipidparticle (e.g., a SNALP formulation) comprising one or more interferingRNAs (e.g., siRNAs) described herein (e.g., one or more siRNAs targetingthe APOB, APOC3, PCSK9, DGAT1, and/or DGAT2 gene). In some embodiments,administration of nucleic acid-lipid particles comprising one or moresiRNAs described herein reduces liver mRNA levels of the target gene(e.g., in a human or in an animal model such as a mouse model or monkeymodel) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any range therein)relative to liver mRNA levels of the target gene detected in the absenceof the siRNA (e.g., buffer control or irrelevant siRNA control). Inother embodiments, administration of nucleic acid-lipid particlescomprising one or more siRNAs described herein reduces liver mRNA levelsof the target gene (e.g., in a human or in an animal model such as amouse model or monkey model) for at least about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 days or more(or any range therein) relative to a negative control such as, e.g., abuffer control or an irrelevant siRNA control.

In certain other aspects, the present invention provides methods fortreating, preventing, reducing the risk or likelihood of developing(e.g., reducing the susceptibility to), delaying the onset of, and/orameliorating one or more symptoms associated with a lipid disorder in amammal (e.g., human) in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of anucleic acid-lipid particle (e.g., a SNALP formulation) comprising oneor more interfering RNA molecules (e.g., siRNAs) described herein (e.g.,one or more siRNAs targeting the APOB, APOC3, PCSK9, DGAT1, and/or DGAT2gene). Non-limiting examples of lipid disorders are described above andinclude dyslipidemia and atherosclerosis.

In a related aspect, the present invention provides a method fortreating and/or ameliorating one or more symptoms associated withatherosclerosis or a dyslipidemia such as hyperlipidemia (e.g., elevatedlevels of triglycerides and/or cholesterol) in a mammal (e.g., human) inneed thereof (e.g., a mammal with atheromatous plaques, elevatedtriglyceride levels, and/or elevated cholesterol levels), the methodcomprising administering to the mammal a therapeutically effectiveamount of a nucleic acid-lipid particle (e.g., a SNALP formulation)comprising one or more interfering RNAs (e.g., siRNAs) described herein(e.g., one or more siRNAs targeting the APOB, APOC3, PCSK9, DGAT1,and/or DGAT2 gene). In some embodiments, administration of nucleicacid-lipid particles comprising one or more siRNA molecules describedherein reduces the level of atherosclerosis (e.g., decreases the sizeand/or number of atheromatous plaques or lesions) or blood (e.g., serumand/or plasma) triglyceride and/or cholesterol levels (e.g., in a humanor in an animal model such as a mouse model or monkey model) by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% (or any range therein) relative tothe level of atherosclerosis, blood triglyceride levels, or bloodcholesterol levels detected in the absence of the siRNA (e.g., buffercontrol or irrelevant siRNA control).

In another related aspect, the present invention provides a method forreducing the risk or likelihood of developing (e.g., reducing thesusceptibility to) atherosclerosis or a dyslipidemia such ashyperlipidemia (e.g., elevated levels of triglycerides and/orcholesterol) in a mammal (e.g., human) at risk of developingatherosclerosis or dyslipidemia, the method comprising administering tothe mammal a therapeutically effective amount of a nucleic acid-lipidparticle (e.g., a SNALP formulation) comprising one or more interferingRNAs (e.g., siRNAs) described herein (e.g., one or more siRNAs targetingthe APOB, APOC3, PCSK9, DGAT1, and/or DGAT2 gene). In some embodiments,administration of nucleic acid-lipid particles comprising one or moresiRNA molecules described herein reduces the risk or likelihood ofdeveloping atherosclerosis or dyslipidemia (e.g., in a human or in ananimal model such as a mouse model or monkey model) by at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% (or any range therein) relative to therisk or likelihood of developing atherosclerosis or dyslipidemia in theabsence of the siRNA (e.g., buffer control or irrelevant siRNA control).

In yet another related aspect, the present invention provides a methodfor preventing or delaying the onset of atherosclerosis or adyslipidemia such as hyperlipidemia (e.g., elevated levels oftriglycerides and/or cholesterol) in a mammal (e.g., human) at risk ofdeveloping atherosclerosis or dyslipidemia, the method comprisingadministering to the mammal a therapeutically effective amount of anucleic acid-lipid particle (e.g., a SNALP formulation) comprising oneor more interfering RNAs (e.g., siRNAs) described herein (e.g., one ormore siRNAs targeting the APOB, APOC3, PCSK9, DGAT1, and/or DGAT2 gene).

In a further related aspect, the present invention provides a method forlowering or reducing cholesterol levels in a mammal (e.g., human) inneed thereof (e.g., a mammal with elevated blood cholesterol levels),the method comprising administering to the mammal a therapeuticallyeffective amount of a nucleic acid-lipid particle (e.g., a SNALPformulation) comprising one or more interfering RNAs (e.g., siRNAs)described herein (e.g., one or more siRNAs targeting the APOB, APOC3,PCSK9, DGAT1, and/or DGAT2 gene). In particular embodiments,administration of nucleic acid-lipid particles (e.g., SNALP) comprisingone or more siRNA molecules described herein lowers or reduces blood(e.g., serum and/or plasma) cholesterol levels. In some embodiments,administration of nucleic acid-lipid particles (e.g., SNALP) comprisingone or more siRNAs described herein reduces blood cholesterol levels(e.g., in a human or in an animal model such as a mouse model or monkeymodel) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (or any rangetherein) relative to blood cholesterol levels detected in the absence ofthe siRNA (e.g., buffer control or irrelevant siRNA control). In certaininstances, administration of nucleic acid-lipid particles (e.g., SNALP)comprising one or more siRNA molecules described herein elevatesHDL-cholesterol levels and/or reduces LDL-cholesterol levels.

In another related aspect, the present invention provides a method forlowering or reducing triglyceride levels in a mammal (e.g., human) inneed thereof (e.g., a mammal with elevated blood triglyceride levels),the method comprising administering to the mammal a therapeuticallyeffective amount of a nucleic acid-lipid particle (e.g., a SNALPformulation) comprising one or more interfering RNAs (e.g., siRNAs)described herein (e.g., one or more siRNAs targeting the APOB, APOC3,PCSK9, DGAT1, and/or DGAT2 gene). In particular embodiments,administration of nucleic acid-lipid particles (e.g., SNALP) comprisingone or more siRNA molecules described herein lowers or reduces blood(e.g., serum and/or plasma) triglyceride levels. In certain embodiments,administration of nucleic acid-lipid particles comprising one or moresiRNA molecules described herein reduces blood triglyceride levels(e.g., in a human or in an animal model such as a mouse model or monkeymodel) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (or any rangetherein) relative to blood triglyceride levels detected in the absenceof the siRNA (e.g., buffer control or irrelevant siRNA control). Inother embodiments, administration of nucleic acid-lipid particles of theinvention lowers or reduces hepatic (i.e., liver) triglyceride levels.

In an additional related aspect, the present invention provides a methodfor lowering or reducing glucose levels in a mammal (e.g., human) inneed thereof (e.g., a mammal with elevated blood glucose levels), themethod comprising administering to the mammal a therapeuticallyeffective amount of a nucleic acid-lipid particle (e.g., a SNALPformulation) comprising one or more interfering RNAs (e.g., siRNAs)described herein (e.g., one or more siRNAs targeting the APOB, APOC3,PCSK9, DGAT1, and/or DGAT2 gene). In particular embodiments,administration of nucleic acid-lipid particles (e.g., SNALP) comprisingone or more siRNAs described herein lowers or reduces blood (e.g., serumand/or plasma) glucose levels. In some embodiments, administration ofnucleic acid-lipid particles comprising one or more siRNAs describedherein reduces blood glucose levels (e.g., in a human or in an animalmodel such as a mouse model or monkey model) by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% (or any range therein) relative to blood glucoselevels detected in the absence of the siRNA (e.g., buffer control orirrelevant siRNA control).

IV. Lipid Particles

The present invention provides lipid particles comprising one or more ofthe cationic (amino) lipids of Formula I-XIV as described herein. Insome embodiments, the lipid particles of the invention further compriseone or more non-cationic lipids. In other embodiments, the lipidparticles further comprise one or more conjugated lipids capable ofreducing or inhibiting particle aggregation. In additional embodiments,the lipid particles further comprise one or more therapeutic nucleicacids (e.g., interfering RNA such as siRNA).

Lipid particles include, but are not limited to, lipid vesicles such asliposomes. As used herein, a lipid vesicle includes a structure havinglipid-containing membranes enclosing an aqueous interior. In particularembodiments, lipid vesicles comprising one or more of the cationiclipids described herein are used to encapsulate nucleic acids within thelipid vesicles. In other embodiments, lipid vesicles comprising one ormore of the cationic lipids described herein are complexed with nucleicacids to form lipoplexes.

The lipid particles of the present invention preferably comprise anactive agent or therapeutic agent such as a therapeutic nucleic acid(e.g., an interfering RNA), a cationic lipid of Formula I-XIV, anon-cationic lipid, and a conjugated lipid that inhibits aggregation ofparticles. In some embodiments, the therapeutic nucleic acid is fullyencapsulated within the lipid portion of the lipid particle such thatthe therapeutic nucleic acid in the lipid particle is resistant inaqueous solution to enzymatic degradation, e.g., by a nuclease orprotease. In other embodiments, the lipid particles described herein aresubstantially non-toxic to mammals such as humans. The lipid particlesof the invention typically have a mean diameter of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, or from about 70 to about 90 nm. The lipid particles ofthe invention also typically have a lipid:therapeutic agent (e.g.,lipid:nucleic acid) ratio (mass/mass ratio) of from about 1:1 to about100:1, from about 1:1 to about 50:1, from about 2:1 to about 25:1, fromabout 3:1 to about 20:1, from about 5:1 to about 15:1, or from about 5:1to about 10:1.

In preferred embodiments, the lipid particles of the invention areserum-stable nucleic acid-lipid particles (SNALP) which comprise aninterfering RNA (e.g., dsRNA such as siRNA, Dicer-substrate dsRNA,shRNA, aiRNA, and/or miRNA), a cationic lipid (e.g., one or morecationic lipids of Formula I-XIV or salts thereof as set forth herein),a non-cationic lipid (e.g., mixtures of one or more phospholipids andcholesterol), and a conjugated lipid that inhibits aggregation of theparticles (e.g., one or more PEG-lipid conjugates). The SNALP maycomprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodifiedand/or modified interfering RNA (e.g., siRNA) that target one or more ofthe genes described herein. Nucleic acid-lipid particles and theirmethod of preparation are described in, e.g., U.S. Pat. Nos. 5,753,613;5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and 6,320,017;and PCT Publication No. WO 96/40964, the disclosures of which are eachherein incorporated by reference in their entirety for all purposes.

In the nucleic acid-lipid particles of the invention, the nucleic acidmay be fully encapsulated within the lipid portion of the particle,thereby protecting the nucleic acid from nuclease degradation. Inpreferred embodiments, a SNALP comprising a nucleic acid such as aninterfering RNA is fully encapsulated within the lipid portion of theparticle, thereby protecting the nucleic acid from nuclease degradation.In certain instances, the nucleic acid in the SNALP is not substantiallydegraded after exposure of the particle to a nuclease at 37° C. for atleast about 20, 30, 45, or 60 minutes. In certain other instances, thenucleic acid in the SNALP is not substantially degraded after incubationof the particle in serum at 37° C. for at least about 30, 45, or 60minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, thenucleic acid is complexed with the lipid portion of the particle. One ofthe benefits of the formulations of the present invention is that thenucleic acid-lipid particle compositions are substantially non-toxic tomammals such as humans.

The term “fully encapsulated” indicates that the nucleic acid in thenucleic acid-lipid particle is not significantly degraded after exposureto serum or a nuclease assay that would significantly degrade free DNAor RNA. In a fully encapsulated system, preferably less than about 25%of the nucleic acid in the particle is degraded in a treatment thatwould normally degrade 100% of free nucleic acid, more preferably lessthan about 10%, and most preferably less than about 5% of the nucleicacid in the particle is degraded. “Fully encapsulated” also indicatesthat the nucleic acid-lipid particles are serum-stable, that is, thatthey do not rapidly decompose into their component parts upon in vivoadministration.

In the context of nucleic acids, full encapsulation may be determined byperforming a membrane-impermeable fluorescent dye exclusion assay, whichuses a dye that has enhanced fluorescence when associated with nucleicacid. Specific dyes such as OliGreen® and RiboGreen® (Invitrogen Corp.;Carlsbad, Calif.) are available for the quantitative determination ofplasmid DNA, single-stranded deoxyribonucleotides, and/or single- ordouble-stranded ribonucleotides. Encapsulation is determined by addingthe dye to a liposomal formulation, measuring the resultingfluorescence, and comparing it to the fluorescence observed uponaddition of a small amount of nonionic detergent. Detergent-mediateddisruption of the liposomal bilayer releases the encapsulated nucleicacid, allowing it to interact with the membrane-impermeable dye. Nucleicacid encapsulation may be calculated as E=(I_(o)−I)/I_(o), where I andI_(o) refer to the fluorescence intensities before and after theaddition of detergent (see, Wheeler et al., Gene Ther., 6:271-281(1999)).

In other embodiments, the present invention provides a nucleicacid-lipid particle (e.g., SNALP) composition comprising a plurality ofnucleic acid-lipid particles.

In some instances, the SNALP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the particles have the nucleic acid encapsulated therein.

In other instances, the SNALP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the input nucleic acid is encapsulated in the particles.

Depending on the intended use of the lipid particles of the invention,the proportions of the components can be varied and the deliveryefficiency of a particular formulation can be measured using, e.g., anendosomal release parameter (ERP) assay.

In particular embodiments, the present invention provides a lipidparticle (e.g., SNALP) composition comprising a plurality of lipidparticles described herein and an antioxidant. In certain instances, theantioxidant in the lipid particle composition reduces, prevents, and/orinhibits the degradation of a cationic lipid present in the lipidparticle. In instances wherein the active agent is a therapeutic nucleicacid such as an interfering RNA (e.g., siRNA), the antioxidant in thelipid particle composition reduces, prevents, and/or inhibits thedegradation of the nucleic acid payload, e.g., by reducing, preventing,and/or inhibiting the formation of adducts between the nucleic acid andthe cationic lipid. Non-limiting examples of antioxidants includehydrophilic antioxidants such as chelating agents (e.g., metal chelatorssuch as ethylenediaminetetraacetic acid (EDTA), citrate, and the like),lipophilic antioxidants (e.g., vitamin E isomers, polyphenols, and thelike), salts thereof; and mixtures thereof. If needed, the antioxidantis typically present in an amount sufficient to prevent, inhibit, and/orreduce the degradation of the cationic lipid and/or active agent presentin the particle, e.g., at least about 20 mM EDTA or a salt thereof, orat least about 100 mM citrate or a salt thereof. An antioxidant such asEDTA and/or citrate may be included at any step or at multiple steps inthe lipid particle formation process described in Section V (e.g., priorto, during, and/or after lipid particle formation).

Additional embodiments related to methods of preventing the degradationof cationic lipids and/or active agents (e.g., therapeutic nucleicacids) present in lipid particles, compositions comprising lipidparticles stabilized by these methods, methods of making these lipidparticles, and methods of delivering and/or administering these lipidparticles are described in U.S. Provisional Application No. 61/265,671,entitled “SNALP Formulations Containing Antioxidants,” filed Dec. 1,2009, the disclosure of which is herein incorporated by reference in itsentirety for all purposes.

A. Therapeutic Nucleic Acids

The lipid particles of the present invention are associated with anucleic acid, resulting in a nucleic acid-lipid particle (e.g., SNALP).In some embodiments, the nucleic acid is fully encapsulated in the lipidparticle. As used herein, the term “nucleic acid” includes anyoligonucleotide or polynucleotide, with fragments containing up to 60nucleotides generally termed oligonucleotides, and longer fragmentstermed polynucleotides. In particular embodiments, oligonucleotides ofthe invention are from about 15 to about 60 nucleotides in length.Nucleic acid may be administered alone in the lipid particles of theinvention, or in combination (e.g., co-administered) with lipidparticles of the invention comprising peptides, polypeptides, or smallmolecules such as conventional drugs. Similarly, when used to treatdiseases and disorders involving hypercholesterolemia, the nucleic acid,such as the interfering RNA, can be administered alone orco-administered (i.e., concurrently or consecutively) with conventionalagents used to treat, e.g., a disease or disorder involvinghypercholesterolemia. Such agents include statins such as, e.g.,Lipitor®, Mevacor®, Zocor®, Lescol®, Crestor®, and Advicor®.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally-occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also include polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake, reduced immunogenicity, and increasedstability in the presence of nucleases.

Oligonucleotides are generally classified as deoxyribooligonucleotidesor ribooligonucleotides. A deoxyribooligonucleotide consists of a5-carbon sugar called deoxyribose joined covalently to phosphate at the5′ and 3′ carbons of this sugar to form an alternating, unbranchedpolymer. A ribooligonucleotide consists of a similar repeating structurewhere the 5-carbon sugar is ribose.

The nucleic acid that is present in a nucleic acid-lipid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. In preferredembodiments, the nucleic acids are double-stranded RNA. Examples ofdouble-stranded RNA are described herein and include, e.g., siRNA andother RNAi agents such as Dicer-substrate dsRNA, shRNA, aiRNA, andpre-miRNA. In other preferred embodiments, the nucleic acids aresingle-stranded nucleic acids. Single-stranded nucleic acids include,e.g., antisense oligonucleotides, ribozymes, mature miRNA, andtriplex-forming oligonucleotides. In further embodiments, the nucleicacids are double-stranded DNA. Examples of double-stranded DNA include,e.g., DNA-DNA hybrids comprising a DNA sense strand and a DNA antisensestrand as described in PCT Publication No. WO 2004/104199, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

Nucleic acids of the invention may be of various lengths, generallydependent upon the particular form of nucleic acid. For example, inparticular embodiments, plasmids or genes may be from about 1,000 toabout 100,000 nucleotide residues in length. In particular embodiments,oligonucleotides may range from about 10 to about 100 nucleotides inlength. In various related embodiments, oligonucleotides, bothsingle-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 60 nucleotides, from about 15 to about 60nucleotides, from about 20 to about 50 nucleotides, from about 15 toabout 30 nucleotides, or from about 20 to about 30 nucleotides inlength.

In particular embodiments, an oligonucleotide (or a strand thereof) ofthe invention specifically hybridizes to or is complementary to a targetpolynucleotide sequence. The terms “specifically hybridizable” and“complementary” as used herein indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. In preferred embodiments,an oligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target sequence interferes with the normalfunction of the target sequence to cause a loss of utility or expressionthere from, and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or, in the case of in vitro assays, under conditions in whichthe assays are conducted. Thus, the oligonucleotide may include 1, 2, 3,or more base substitutions as compared to the region of a gene or mRNAsequence that it is targeting or to which it specifically hybridizes.

a) siRNA

The siRNA component of the nucleic acid-lipid particles of the presentinvention is capable of silencing the expression of a target gene ofinterest, such as APOB, APOC3, PCSK9, DGAT1, DGAT2, or combinationsthereof. Each strand of the siRNA duplex is typically about 15 to about60 nucleotides in length, preferably about 15 to about 30 nucleotides inlength. In certain embodiments, the siRNA comprises at least onemodified nucleotide. The modified siRNA is generally lessimmunostimulatory than a corresponding unmodified siRNA sequence andretains RNAi activity against the target gene of interest. In someembodiments, the modified siRNA contains at least one 2′OMe purine orpyrimidine nucleotide such as a 2′OMe-guanosine, 2′OMe-uridine,2′OMe-adenosine, and/or 2′OMe-cytosine nucleotide. The modifiednucleotides can be present in one strand (i.e., sense or antisense) orboth strands of the siRNA. In some preferred embodiments, one or more ofthe uridine and/or guanosine nucleotides are modified (e.g.,2′OMe-modified) in one strand (i.e., sense or antisense) or both strandsof the siRNA. In these embodiments, the modified siRNA can furthercomprise one or more modified (e.g., 2′OMe-modified) adenosine and/ormodified (e.g., 2′OMe-modified) cytosine nucleotides. In other preferredembodiments, only uridine and/or guanosine nucleotides are modified(e.g., 2′OMe-modified) in one strand (i.e., sense or antisense) or bothstrands of the siRNA. The siRNA sequences may have overhangs (e.g., 3′or 5′ overhangs as described in Elbashir et al., Genes Dev., 15:188(2001) or Nykänen et al., Cell, 107:309 (2001)), or may lack overhangs(i.e., have blunt ends).

In particular embodiments, the selective incorporation of modifiednucleotides such as 2′OMe uridine and/or guanosine nucleotides into thedouble-stranded region of either or both strands of the siRNA reduces orcompletely abrogates the immune response to that siRNA molecule. Incertain instances, the immunostimulatory properties of specific siRNAsequences and their ability to silence gene expression can be balancedor optimized by the introduction of minimal and selective 2′OMemodifications within the double-stranded region of the siRNA duplex.This can be achieved at therapeutically viable siRNA doses withoutcytokine induction, toxicity, and off-target effects associated with theuse of unmodified siRNA.

The modified siRNA generally comprises from about 1% to about 100%(e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%) modified nucleotides in the double-stranded region ofthe siRNA duplex. In certain embodiments, one, two, three, four, five,six, seven, eight, nine, ten, or more of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides. Incertain other embodiments, some or all of the modified nucleotides inthe double-stranded region of the siRNA are 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more nucleotides apart from each other. In one preferredembodiment, none of the modified nucleotides in the double-strandedregion of the siRNA are adjacent to each other (e.g., there is a gap ofat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified nucleotides betweeneach modified nucleotide). In another preferred embodiment, at least twoof the modified nucleotides in the double-stranded region of the siRNAare adjacent to each other (e.g., there are no unmodified nucleotidesbetween two or more modified nucleotides). In other preferredembodiments, at least three, at least four, or at least five of themodified nucleotides in the double-stranded region of the siRNA areadjacent to each other.

In some embodiments, less than about 50% (e.g., less than about 49%,48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, or 36%,preferably less than about 35%, 34%, 33%, 32%, 31%, or 30%) of thenucleotides in the double-stranded region of the siRNA comprise modified(e.g., 2′OMe) nucleotides. In one aspect of these embodiments, less thanabout 50% of the uridine and/or guanosine nucleotides in thedouble-stranded region of one or both strands of the siRNA areselectively (e.g., only) modified. In another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the siRNA,wherein the siRNA comprises at least one 2′OMe-guanosine nucleotide andat least one 2′OMe-uridine nucleotide, and wherein 2′OMe-guanosinenucleotides and 2′OMe-uridine nucleotides are the only 2′OMe nucleotidespresent in the double-stranded region. In yet another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the siRNA does not comprise 2′OMe-cytosine nucleotides in thedouble-stranded region. In a further aspect of these embodiments, lessthan about 50% of the nucleotides in the double-stranded region of thesiRNA comprise 2′OMe nucleotides, wherein the siRNA comprises 2′OMenucleotides in both strands of the siRNA, wherein the siRNA comprises atleast one 2′OMe-guanosine nucleotide and at least one 2′OMe-uridinenucleotide, and wherein the siRNA does not comprise 2′OMe-cytosinenucleotides in the double-stranded region. In another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the 2′OMe nucleotides in the double-stranded region are notadjacent to each other.

In other embodiments, from about 1% to about 50% (e.g., from about5%-50%, 10%-50%, 15%-50%, 20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%,45%-50%, 5%-45%, 10%-45%, 15%-45%; 20%-45%, 25%-45%, 30%-45%, 35%-45%,40%-45%, 5%-40%, 10%-40%, 15%-40%, 20%-40%, 25%-40%, 25%-39%, 25%-38%,25%-37%, 25%-36%, 26%-39%, 26%-38%, 26%-37%, 26%-36%, 27%-39%, 27%-38%,27%-37%, 27%-36%, 28%-39%, 28%-38%, 28%-37%, 28%-36%, 29%-39%, 29%-38%,29%-37%, 29%-36%, 30%-40%, 30%-39%, 30%-38%, 30%-37%, 30%-36%, 31%-39%,31%-38%, 31%-37%, 31%-36%, 32%-39%, 32%-38%, 32%-37%, 32%-36%, 33%-39%,33%-38%, 33%-37%, 33%-36%, 34%-39%, 34%-38%, 34%-37%, 34%-36%, 35%-40%,5%-35%, 10%-35%, 15%-35%, 20%-35%, 21%-35%, 22%-35%, 23%-35%, 24%-35%,25%-35%, 26%-35%, 27%-35%, 28%-35%, 29%-35%, 30%-35%, 31%-35%, 32%-35%,33%-35%, 34%-35%, 30%-34%, 31%-34%, 32%-34%, 33%-34%, 30%-33%, 31%-33%,32%-33%, 30%-32%, 31%-32%, 25%-34%, 25%-33%, 25%-32%, 25%-31%, 26%-34%,26%-33%, 26%-32%, 26%-31%, 27%-34%, 27%-33%, 27%-32%, 27%-31%, 28%-34%,28%-33%, 28%-32%, 28%-31%, 29%-34%, 29%-33%, 29%-32%, 29%-31%, 5%-30%,10%-30%, 15%-30%, 20%-34%, 20%-33%, 20%-32%, 20%-31%, 20%-30%, 21%-30%,22%-30%, 23%-30%, 24%-30%, 25%-30%, 25%-29%, 25%-28%, 25%-27%, 25%-26%,26%-30%, 26%-29%, 26%-28%, 26%-27%, 27%-30%, 27%-29%, 27%-28%, 28%-30%,28%-29%, 29%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-29%, 20%-28%, 20%-27%,20%-26%, 20%-25%, 5%-20%, 10%-20%, 15%-20%, 5%-15%, 10%-15%, or 5%-10%)of the nucleotides in the double-stranded region of the siRNA comprisemodified nucleotides. In one aspect of these embodiments, from about 1%to about 50% of the uridine and/or guanosine nucleotides in thedouble-stranded region of one or both strands of the siRNA areselectively (e.g., only) modified. In another aspect of theseembodiments, from about 1% to about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the siRNA,wherein the siRNA comprises at least one 2′OMe-guanosine nucleotide andat least one 2′OMe-uridine nucleotide, and wherein 2′OMe-guanosinenucleotides and 2′OMe-uridine nucleotides are the only 2′OMe nucleotidespresent in the double-stranded region. In yet another aspect of theseembodiments, from about 1% to about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the siRNA does not comprise 2′OMe-cytosine nucleotides in thedouble-stranded region. In a further aspect of these embodiments, fromabout 1% to about 50% of the nucleotides in the double-stranded regionof the siRNA comprise 2′OMe nucleotides, wherein the siRNA comprises2′OMe nucleotides in both strands of the siRNA, wherein the siRNAcomprises at least one 2′OMe-guanosine nucleotide and at least one2′OMe-uridine nucleotide, and wherein the siRNA does not comprise2′OMe-cytosine nucleotides in the double-stranded region. In anotheraspect of these embodiments, from about 1% to about 50% of thenucleotides in the double-stranded region of the siRNA comprise 2′OMenucleotides, wherein the siRNA comprises 2′OMe nucleotides in bothstrands of the modified siRNA, wherein the siRNA comprises 2′OMenucleotides selected from the group consisting of 2′OMe-guanosinenucleotides, 2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, andmixtures thereof, and wherein the 2′OMe nucleotides in thedouble-stranded region are not adjacent to each other.

In certain embodiments, the siRNA component of the nucleic acid-lipidparticles of the present invention (e.g., SNALP) comprises an asymmetricsiRNA duplex as described in PCT Publication No. WO 2004/078941, whichcomprises a double-stranded region consisting of a DNA sense strand andan RNA antisense strand (e.g., a DNA-RNA hybrid), wherein a blockingagent is located on the siRNA duplex. In some instances, the asymmetricsiRNA duplex can be chemically modified as described herein. Othernon-limiting examples of asymmetric siRNA duplexes are described in PCTPublication No. WO 2006/074108, which discloses self-protectedoligonucleotides comprising a region having a sequence complementary toone, two, three, or more same or different target mRNA sequences (e.g.,multivalent siRNAs) and one or more self-complementary regions. Yetother non-limiting examples of asymmetric siRNA duplexes are describedin PCT Publication No. WO 2009/076321, which discloses self-formingasymmetric precursor polynucleotides comprising a targeting regioncomprising a polynucleotide sequence complementary to a region of one,two, three, or more same or different target mRNA sequences (e.g.,multivalent siRNAs); a first self-complementary region; and a secondself-complementary region, wherein the first and secondself-complementary regions are located one at each end of the targetingregion and both self-complementary regions form stem-loop structures,wherein the first self-complementary region is capable of being cleavedby a RNase III endoribonuclease that is not a class IV DICERendoribonuclease, and wherein both self-complementary regions comprise anucleotide sequence that is complementary to a region of the target genesequence, but wherein a portion of the target sequence present in thetargeting region does not have a complementary sequence in either of theself-complementary regions. The disclosures of each of the above patentdocuments are herein incorporated by reference in their entirety for allpurposes.

Additional ranges, percentages, and patterns of modifications that maybe introduced into siRNA are described in U.S. Patent Publication No.20070135372, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

(1) Selection of siRNA Sequences

Suitable siRNA sequences can be identified using any means known in theart. Typically, the methods described in Elbashir et al., Nature,411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) arecombined with rational design rules set forth in Reynolds et al., NatureBiotech., 22(3):326-330 (2004).

As a non-limiting example, the nucleotide sequence 3′ of the AUG startcodon of a transcript from the target gene of interest may be scannedfor dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, wherein N=C, G,or U) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). Thenucleotides immediately 3′ to the dinucleotide sequences are identifiedas potential siRNA sequences (i.e., a target sequence or a sense strandsequence). Typically, the 19, 21, 23, 25, 27, 29, 31, 33, 35, or morenucleotides immediately 3′ to the dinucleotide sequences are identifiedas potential siRNA sequences. In some embodiments, the dinucleotidesequence is an AA or NA sequence and the 19 nucleotides immediately 3′to the AA or NA dinucleotide are identified as potential siRNAsequences. siRNA sequences are usually spaced at different positionsalong the length of the target gene. To further enhance silencingefficiency of the siRNA sequences, potential siRNA sequences may beanalyzed to identify sites that do not contain regions of homology toother coding sequences, e.g., in the target cell or organism. Forexample, a suitable siRNA sequence of about 21 base pairs typically willnot have more than 16-17 contiguous base pairs of homology to codingsequences in the target cell or organism. If the siRNA sequences are tobe expressed from an RNA Pol III promoter, siRNA sequences lacking morethan 4 contiguous A's or T's are selected.

Once a potential siRNA sequence has been identified, a complementarysequence (i.e., an antisense strand sequence) can be designed. Apotential siRNA sequence can also be analyzed using a variety ofcriteria known in the art. For example, to enhance their silencingefficiency, the siRNA sequences may be analyzed by a rational designalgorithm to identify sequences that have one or more of the followingfeatures: (1) G/C content of about 25% to about 60% G/C; (2) at least 3A/Us at positions 15-19 of the sense strand; (3) no internal repeats;(4) an A at position 19 of the sense strand; (5) an A at position 3 ofthe sense strand; (6) a U at position 10 of the sense strand; (7) no G/Cat position 19 of the sense strand; and (8) no G at position 13 of thesense strand. siRNA design tools that incorporate algorithms that assignsuitable values of each of these features and are useful for selectionof siRNA can be found at, e.g.,http://ihome.ust.hk/˜bokcmho/siRNA/siRNA.html. One of skill in the artwill appreciate that sequences with one or more of the foregoingcharacteristics may be selected for further analysis and testing aspotential siRNA sequences.

Additionally, potential siRNA sequences with one or more of thefollowing criteria can often be eliminated as siRNA: (1) sequencescomprising a stretch of 4 or more of the same base in a row; (2)sequences comprising homopolymers of Gs (i.e., to reduce possiblenon-specific effects due to structural characteristics of thesepolymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC,AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in arow; and (5) sequences comprising direct repeats of 4 or more baseswithin the candidates resulting in internal fold-back structures.However, one of skill in the art will appreciate that sequences with oneor more of the foregoing characteristics may still be selected forfurther analysis and testing as potential siRNA sequences.

In some embodiments, potential siRNA sequences may be further analyzedbased on siRNA duplex asymmetry as described in, e.g., Khvorova et al.,Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208 (2003).In other embodiments, potential siRNA sequences may be further analyzedbased on secondary structure at the target site as described in, e.g.,Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example,secondary structure at the target site can be modeled using the Mfoldalgorithm (available at http://mfold.burnet.edu.au/rna_form) to selectsiRNA sequences which favor accessibility at the target site where lesssecondary structure in the form of base-pairing and stem-loops ispresent.

Once a potential siRNA sequence has been identified, the sequence can beanalyzed for the presence of any immunostimulatory properties, e.g.,using an in vitro cytokine assay or an in vivo animal model. Motifs inthe sense and/or antisense strand of the siRNA sequence such as GU-richmotifs (e.g., 5′-GU-3′,5′-UGU-3′,5′-GUGU-3′,5′-UGUGU-3′, etc.) can alsoprovide an indication of whether the sequence may be immunostimulatory.Once an siRNA molecule is found to be immunostimulatory, it can then bemodified to decrease its immunostimulatory properties as describedherein. As a non-limiting example, an siRNA sequence can be contactedwith a mammalian responder cell under conditions such that the cellproduces a detectable immune response to determine whether the siRNA isan immunostimulatory or a non-immunostimulatory siRNA. The mammalianresponder cell may be from a naïve mammal (i.e., a mammal that has notpreviously been in contact with the gene product of the siRNA sequence).The mammalian responder cell may be, e.g., a peripheral bloodmononuclear cell (PBMC), a macrophage, and the like. The detectableimmune response may comprise production of a cytokine or growth factorsuch as, e.g., TNF-α, IFN-α, IFN-β, IFN-γ, IL-6, IL-8, IL-12, or acombination thereof. An siRNA molecule identified as beingimmunostimulatory can then be modified to decrease its immunostimulatoryproperties by replacing at least one of the nucleotides on the senseand/or antisense strand with modified nucleotides. For example, lessthan about 30% (e.g., less than about 30%, 25%, 20%, 15%, 10%, or 5%) ofthe nucleotides in the double-stranded region of the siRNA duplex can bereplaced with modified nucleotides such as 2′OMe nucleotides. Themodified siRNA can then be contacted with a mammalian responder cell asdescribed above to confirm that its immunostimulatory properties havebeen reduced or abrogated.

Suitable in vitro assays for detecting an immune response include, butare not limited to, the double monoclonal antibody sandwich immunoassaytechnique of David et al. (U.S. Pat. No. 4,376,110);monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkhamand Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone,Edinburgh (1970)); the “Western blot” method of Gordon et al. (U.S. Pat.No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assays(ELISA) as described, for example, by Raines et al., J. Biol. Chem.,257:5154-5160 (1982); immunocytochemical techniques, including the useof fluorochromes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980));and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad.Sci. USA, 81:2396-2400 (1984)). In addition to the immunoassaysdescribed above, a number of other immunoassays are available, includingthose described in U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Thedisclosures of these references are herein incorporated by reference intheir entirety for all purposes.

A non-limiting example of an in vivo model for detecting an immuneresponse includes an in vivo mouse cytokine induction assay as describedin, e.g., Judge et al., Mol. Ther., 13:494-505 (2006). In certainembodiments, the assay that can be performed as follows: (1) siRNA canbe administered by standard intravenous injection in the lateral tailvein; (2) blood can be collected by cardiac puncture about 6 hours afteradministration and processed as plasma for cytokine analysis; and (3)cytokines can be quantified using sandwich ELISA kits according to themanufacturer's instructions (e.g., mouse and human IFN-α (PBLBiomedical; Piscataway, N.J.); human IL-6 and TNF-α (eBioscience; SanDiego, Calif.); and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; SanDiego, Calif.)).

Monoclonal antibodies that specifically bind cytokines and growthfactors are commercially available from multiple sources and can begenerated using methods known in the art (see, e.g., Kohler et al.,Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)).Generation of monoclonal antibodies has been previously described andcan be accomplished by any means known in the art (Buhring et al., inHybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, themonoclonal antibody is labeled (e.g., with any composition detectable byspectroscopic, photochemical, biochemical, electrical, optical, orchemical means) to facilitate detection.

(2) Generating siRNA Molecules

siRNA can be provided in several forms including, e.g., as one or moreisolated small-interfering RNA (siRNA) duplexes, as longerdouble-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from atranscriptional cassette in a DNA plasmid. In some embodiments, siRNAmay be produced enzymatically or by partial/total organic synthesis, andmodified ribonucleotides can be introduced by in vitro enzymatic ororganic synthesis. In certain instances, each strand is preparedchemically. Methods of synthesizing RNA molecules are known in the art,e.g., the chemical synthesis methods as described in Verma and Eckstein(1998) or as described herein.

An RNA population can be used to provide long precursor RNAs, or longprecursor RNAs that have substantial or complete identity to a selectedtarget sequence can be used to make the siRNA. The RNAs can be isolatedfrom cells or tissue, synthesized, and/or cloned according to methodswell known to those of skill in the art. The RNA can be a mixedpopulation (obtained from cells or tissue, transcribed from cDNA,subtracted, selected, etc.), or can represent a single target sequence.RNA can be naturally occurring (e.g., isolated from tissue or cellsamples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCRproducts or a cloned cDNA), or chemically synthesized.

To form a long dsRNA, for synthetic RNAs, the complement is alsotranscribed in vitro and hybridized to form a dsRNA. If a naturallyoccurring RNA population is used, the RNA complements are also provided(e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g.,by transcribing cDNAs corresponding to the RNA population, or by usingRNA polymerases. The precursor RNAs are then hybridized to form doublestranded RNAs for digestion. The dsRNAs can be directly administered toa subject or can be digested in vitro prior to administration.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see,U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Expressionlibraries are also well known to those of skill in the art. Additionalbasic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994). The disclosures of these references are herein incorporatedby reference in their entirety for all purposes.

Preferably, siRNA are chemically synthesized. The oligonucleotides thatcomprise the siRNA molecules of the invention can be synthesized usingany of a variety of techniques known in the art, such as those describedin Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al.,Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res.,23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59(1997). The synthesis of oligonucleotides makes use of common nucleicacid protecting and coupling groups, such as dimethoxytrityl at the5′-end and phosphoramidites at the 3′-end. As a non-limiting example,small scale syntheses can be conducted on an Applied Biosystemssynthesizer using a 0.2 μmol scale protocol. Alternatively, syntheses atthe 0.2 μmol scale can be performed on a 96-well plate synthesizer fromProtogene (Palo Alto, Calif.). However, a larger or smaller scale ofsynthesis is also within the scope of this invention. Suitable reagentsfor oligonucleotide synthesis, methods for RNA deprotection, and methodsfor RNA purification are known to those of skill in the art.

siRNA molecules can also be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousoligonucleotide fragment or strand separated by a cleavable linker thatis subsequently cleaved to provide separate fragments or strands thathybridize to form the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker. The tandem synthesis of siRNA can bereadily adapted to both multiwell/multiplate synthesis platforms as wellas large scale synthesis platforms employing batch reactors, synthesiscolumns, and the like. Alternatively, siRNA molecules can be assembledfrom two distinct oligonucleotides, wherein one oligonucleotidecomprises the sense strand and the other comprises the antisense strandof the siRNA. For example, each strand can be synthesized separately andjoined together by hybridization or ligation following synthesis and/ordeprotection. In certain other instances, siRNA molecules can besynthesized as a single continuous oligonucleotide fragment, where theself-complementary sense and antisense regions hybridize to form ansiRNA duplex having hairpin secondary structure.

(3) Modifying siRNA Sequences

In certain aspects, siRNA molecules comprise a duplex having two strandsand at least one modified nucleotide in the double-stranded region,wherein each strand is about 15 to about 60 nucleotides in length.Advantageously, the modified siRNA is less immunostimulatory than acorresponding unmodified siRNA sequence, but retains the capability ofsilencing the expression of a target sequence. In preferred embodiments,the degree of chemical modifications introduced into the siRNA strikes abalance between reduction or abrogation of the immunostimulatoryproperties of the siRNA and retention of RNAi activity. As anon-limiting example, an siRNA molecule that targets a gene of interestcan be minimally modified (e.g., less than about 30%, 25%, 20%, 15%,10%, or 5% modified) at selective uridine and/or guanosine nucleotideswithin the siRNA duplex to eliminate the immune response generated bythe siRNA while retaining its capability to silence target geneexpression.

Examples of modified nucleotides suitable for use in the inventioninclude, but are not limited to, ribonucleotides having a 2′-O-methyl(2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl,2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.Modified nucleotides having a Northern conformation such as thosedescribed in, e.g., Saenger, Principles of Nucleic Acid Structure,Springer-Verlag Ed. (1984), are also suitable for use in siRNAmolecules. Such modified nucleotides include, without limitation, lockednucleic acid (LNA) nucleotides (e.g., 2′-O,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl)(MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro (2′Cl) nucleotides, and 2′-azidonucleotides. In certain instances, the siRNA molecules described hereininclude one or more G-clamp nucleotides. A G-clamp nucleotide refers toa modified cytosine analog wherein the modifications confer the abilityto hydrogen bond both Watson-Crick and Hoogsteen faces of acomplementary guanine nucleotide within a duplex (see, e.g., Lin et al.,J. Am. Chem. Soc., 120:8531-8532 (1998)). In addition, nucleotideshaving a nucleotide base analog such as, for example, C-phenyl,C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, andnitroazole derivatives such as 3-nitropyrrole, 4-nitroindole,5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res.,29:2437-2447 (2001)) can be incorporated into siRNA molecules.

In certain embodiments, siRNA molecules may further comprise one or morechemical modifications such as terminal cap moieties, phosphate backbonemodifications, and the like. Examples of terminal cap moieties include,without limitation, inverted deoxy abasic residues, glycerylmodifications, 4′,5′-methylene nucleotides, 1-(β-D-erythrofuranosyl)nucleotides, 4′-thio nucleotides, carbocyclic nucleotides,1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modifiedbase nucleotides, threo-pentofuranosyl nucleotides, acyclic 3′,4′-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties,3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties,3′-2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties,5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties,5′-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropylphosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate,hydroxypropyl phosphate, 1,4-butanediol phosphate, 3′-phosphoramidate,5′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate,and bridging or non-bridging methylphosphonate or 5′-mercapto moieties(see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron49:1925 (1993)). Non-limiting examples of phosphate backbonemodifications (i.e., resulting in modified internucleotide linkages)include phosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate, carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker etal., Nucleic Acid Analogues: Synthesis and Properties, in ModernSynthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39 (1994)). Such chemical modifications canoccur at the 5′-end and/or 3′-end of the sense strand, antisense strand,or both strands of the siRNA. The disclosures of these references areherein incorporated by reference in their entirety for all purposes.

In some embodiments, the sense and/or antisense strand of the siRNAmolecule can further comprise a 3′-terminal overhang having about 1 toabout 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides, modified (e.g.,2′OMe) and/or unmodified uridine ribonucleotides, and/or any othercombination of modified (e.g., 2′OMe) and unmodified nucleotides.

Additional examples of modified nucleotides and types of chemicalmodifications that can be introduced into siRNA molecules are described,e.g., in UK Patent No. GB 2,397,818 B and U.S. Patent Publication Nos.20040192626, 20050282188, and 20070135372, the disclosures of which areherein incorporated by reference in their entirety for all purposes.

The siRNA molecules described herein can optionally comprise one or morenon-nucleotides in one or both strands of the siRNA. As used herein, theterm “non-nucleotide” refers to any group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including sugar and/or phosphate substitutions, andallows the remaining bases to exhibit their activity. The group orcompound is abasic in that it does not contain a commonly recognizednucleotide base such as adenosine, guanine, cytosine, uracil, or thymineand therefore lacks a base at the 1′-position.

In other embodiments, chemical modification of the siRNA comprisesattaching a conjugate to the siRNA molecule. The conjugate can beattached at the 5′ and/or 3′-end of the sense and/or antisense strand ofthe siRNA via a covalent attachment such as, e.g., a biodegradablelinker. The conjugate can also be attached to the siRNA, e.g., through acarbamate group or other linking group (see, e.g., U.S. PatentPublication Nos. 20050074771, 20050043219, and 20050158727). In certaininstances, the conjugate is a molecule that facilitates the delivery ofthe siRNA into a cell. Examples of conjugate molecules suitable forattachment to siRNA include, without limitation, steroids such ascholesterol, glycols such as polyethylene glycol (PEG), human serumalbumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates(e.g., folic acid, folate analogs and derivatives thereof), sugars(e.g., galactose, galactosamine, N-acetyl galactosamine, glucose,mannose, fructose, fucose, etc.), phospholipids, peptides, ligands forcellular receptors capable of mediating cellular uptake, andcombinations thereof (see, e.g., U.S. Patent Publication Nos.20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423).Other examples include the lipophilic moiety, vitamin, polymer, peptide,protein, nucleic acid, small molecule, oligosaccharide, carbohydratecluster, intercalator, minor groove binder, cleaving agent, andcross-linking agent conjugate molecules described in U.S. PatentPublication Nos. 20050119470 and 20050107325. Yet other examples includethe 2′-O-alkyl amine, 2′-β-alkoxyalkyl amine, polyamine, C5-cationicmodified pyrimidine, cationic peptide, guanidinium group, amidininiumgroup, cationic amino acid conjugate molecules described in U.S. PatentPublication No. 20050153337. Additional examples include the hydrophobicgroup, membrane active compound, cell penetrating compound, celltargeting signal, interaction modifier, and steric stabilizer conjugatemolecules described in U.S. Patent Publication No. 20040167090. Furtherexamples include the conjugate molecules described in U.S. PatentPublication No. 20050239739. The type of conjugate used and the extentof conjugation to the siRNA molecule can be evaluated for improvedpharmacokinetic profiles, bioavailability, and/or stability of the siRNAwhile retaining RNAi activity. As such, one skilled in the art canscreen siRNA molecules having various conjugates attached thereto toidentify ones having improved properties and full RNAi activity usingany of a variety of well-known in vitro cell culture or in vivo animalmodels. The disclosures of the above-described patent documents areherein incorporated by reference in their entirety for all purposes.

(4) Target Genes

The siRNA component of the nucleic acid-lipid particles of the presentinvention (e.g., SNALP) can be used to downregulate or silence thetranslation (i.e., expression) of a gene of interest. As previouslymentioned, the present invention is based, in part, on the discoverythat the use of certain cationic (amino) lipids in nucleic acid-lipidparticles provide advantages when the particles are used for the in vivodelivery of therapeutic nucleic acids, such as siRNA, into the liver ofa mammal. In particular, it has been unexpectedly found that the nucleicacid-lipid particles of the present invention (i.e., SNALP formulations)containing at least one cationic lipid of Formula I-XIV and at least oneinterfering RNA as disclosed herein show increased potency (i.e.,increased silencing) and/or increased tolerability (e.g., decreasedtoxicity) when targeting a gene of interest in the liver, such as APOB,when compared to other nucleic acid-lipid particle compositionspreviously described. As such, genes of interest include, but are notlimited to, genes associated with metabolic diseases and disorders(e.g., liver diseases and disorders).

Genes associated with metabolic diseases and disorders (e.g., disordersin which the liver is the target and liver diseases and disorders)include, but are not limited to, genes expressed in dyslipidemia, suchas, e.g., apolipoprotein B (APOB) (Genbank Accession No. NM_(—)000384),apolipoprotein CIII (APOC3) (Genbank Accession Nos. NM_(—)000040 andNG_(—)008949 REGION: 5001.8164), apolipoprotein E (APOE) (GenbankAccession Nos. NM_(—)000041 and NG_(—)007084 REGION: 5001.8612),proprotein convertase subtilisin/kexin type 9 (PCSK9) (Genbank AccessionNo. NM_(—)174936), diacylglycerol O-acyltransferase type 1 (DGAT1)(Genbank Accession No. NM_(—)012079), diacylglycerol O-acyltransferasetype 2 (DGAT2) (Genbank Accession No. NM_(—)032564), liver X receptorssuch as LXRα and LXRβ (Genback Accession No. NM_(—)007121), farnesoid Xreceptors (FXR) (Genbank Accession No. NM_(—)005123), sterol-regulatoryelement binding protein (SREBP), site-1 protease (S1P),3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG coenzyme-Areductase); and genes expressed in diabetes, such as, e.g., glucose6-phosphatase (see, e.g., Forman et al., Cell, 81:687 (1995); Seol etal., Mol. Endocrinol., 9:72 (1995), Zavacki et al., Proc. Natl. Acad.Sci. USA, 94:7909 (1997); Sakai et al., Cell, 85:1037-1046 (1996);Duncan et al., J. Biol. Chem., 272:12778-12785 (1997); Willy et al.,Genes Dev., 9:1033-1045 (1995); Lehmann et al., J. Biol. Chem.,272:3137-3140 (1997); Janowski et al., Nature, 383:728-731 (1996); andPeet et al., Cell, 93:693-704 (1998)).

One of skill in the art will appreciate that genes associated withmetabolic diseases and disorders (e.g., diseases and disorders in whichthe liver is a target and liver diseases and disorders) include genesthat are expressed in the liver itself as well as and genes expressed inother organs and tissues. Silencing of sequences that encode genesassociated with metabolic diseases and disorders can conveniently beused in combination with the administration of conventional agents usedto treat the disease or disorder.

In a presently preferred embodiment, the SNALP formulations of thepresent invention are used to deliver to the liver an siRNA moleculethat silences APOB gene expression. Non-limiting examples of siRNAmolecules targeting the APOB gene include, but are not limited to, thosedescribed in U.S. Patent Publication Nos. 20060134189, 20060105976, and20070135372, and PCT Publication No. WO 04/091515, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. In another preferred embodiment, the SNALP formulations of thepresent invention are used to deliver to the liver an siRNA moleculesthat silences APOC3 gene expression. Non-limiting examples of siRNAmolecules targeting the APOC3 gene include, but are not limited to,those described in PCT Application No: PCT/CA2010/000120, filed Jan. 26,2010, the disclosure of which is herein incorporated by reference in itsentirety for all purposes. In yet another preferred embodiment, theSNALP formulations of the present invention are used to deliver to theliver an siRNA molecule that silences PCSK9 gene expression.Non-limiting examples of siRNA molecules targeting the PCSK9 geneinclude those described in U.S. Patent Publication Nos. 20070173473,20080113930, and 20080306015, the disclosures of which are hereinincorporated by reference in their entirety for all purposes. In stillanother preferred embodiment, the SNALP formulations of the presentinvention are used to deliver to the liver siRNA molecules that silenceDGAT1 and/or DGAT2 gene expression. Exemplary siRNA molecules targetingthe DGAT1 gene may be designed using the antisense compounds describedin U.S. Patent Publication No. 20040185559, the disclosure of which isherein incorporated by reference in its entirety for all purposes.Exemplary siRNA molecules targeting the DGAT2 gene may be designed usingthe antisense compounds described in U.S. Patent Publication No.20050043524, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

In addition to being particularly useful for silencing any of APOB,APOC3, PCSK9, DGAT1 and DGAT2, either alone or in various combinations,the SNALP formulations of the present invention are also useful fortreating hepatitis. Exemplary hepatitis virus nucleic acid sequencesthat can be silenced include, but are not limited to, nucleic acidsequences involved in transcription and translation (e.g., En1, En2, X,P) and nucleic acid sequences encoding structural proteins (e.g., coreproteins including C and C-related proteins, capsid and envelopeproteins including S, M, and/or L proteins, or fragments thereof) (see,e.g., FIELDS VIROLOGY, supra). Exemplary Hepatitis C virus (HCV) nucleicacid sequences that can be silenced include, but are not limited to, the5′-untranslated region (5′-UTR), the 3′-untranslated region (3′-UTR),the polyprotein translation initiation codon region, the internalribosome entry site (IRES) sequence, and/or nucleic acid sequencesencoding the core protein, the E1 protein, the E2 protein, the p7protein, the NS2 protein, the NS3 protease/helicase, the NS4A protein,the NS4B protein, the NS5A protein, and/or the NS5B RNA-dependent RNApolymerase. HCV genome sequences are set forth in, e.g., GenbankAccession Nos. NC_(—)004102 (HCV genotype 1a), AJ238799 (HCV genotype1b), NC_(—)009823 (HCV genotype 2), NC_(—)009824 (HCV genotype 3),NC_(—)009825 (HCV genotype 4), NC_(—)009826 (HCV genotype 5), andNC_(—)009827 (HCV genotype 6). Hepatitis A virus nucleic acid sequencesare set forth in, e.g., Genbank Accession No. NC_(—)001489; Hepatitis Bvirus nucleic acid sequences are set forth in, e.g., Genbank AccessionNo. NC_(—)003977; Hepatitis D virus nucleic acid sequence are set forthin, e.g., Genbank Accession No. NC_(—)001653; Hepatitis E virus nucleicacid sequences are set forth in, e.g., Genbank Accession No.NC_(—)001434; and Hepatitis G virus nucleic acid sequences are set forthin, e.g., Genbank Accession No. NC_(—)001710. Silencing of sequencesthat encode genes associated with viral infection and survival canconveniently be used in combination with the administration ofconventional agents used to treat the viral condition. Non-limitingexamples of siRNA molecules targeting hepatitis virus nucleic acidsequences include, but are not limited to, those described in U.S.Patent Publication Nos. 20060281175, 20050058982, and 20070149470; U.S.Pat. No. 7,348,314; and PCT Application No. PCT/CA2010/000444, entitled“Compositions and Methods for Silencing Hepatitis C Virus Expression,”filed Mar. 19, 2010, bearing Attorney Docket No. 020801-008910PC, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

In addition to its utility in silencing the expression of any of theabove-described genes for therapeutic purposes, the siRNA describedherein are also useful in research and development applications as wellas diagnostic, prophylactic, prognostic, clinical, and other healthcareapplications. As a non-limiting example, the siRNA can be used in targetvalidation studies directed at testing whether a gene of interest hasthe potential to be a therapeutic target. The siRNA can also be used intarget identification studies aimed at discovering genes as potentialtherapeutic targets.

(5) Exemplary siRNA Embodiments

In some embodiments, each strand of the siRNA molecule comprises fromabout 15 to about 60 nucleotides in length (e.g., about 15-60, 15-50,15-40, 15-30, 15-25, or 19-25 nucleotides in length, or 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In one particularembodiment, the siRNA is chemically synthesized. The siRNA molecules ofthe invention are capable of silencing the expression of a targetsequence in vitro and/or in vivo.

In other embodiments, the siRNA comprises at least one modifiednucleotide. In certain embodiments, the siRNA comprises one, two, three,four, five, six, seven, eight, nine, ten, or more modified nucleotidesin the double-stranded region. In particular embodiments, less thanabout 50% (e.g., less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, or 5%) of the nucleotides in the double-stranded region of thesiRNA comprise modified nucleotides. In preferred embodiments, fromabout 1% to about 50% (e.g., from about 5%-50%, 10%-50%, 15%-50%,20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%, 45%-50%, 5%-45%, 10%-45%,15%-45%, 20%-45%, 25%-45%, 30%-45%, 35%-45%, 40%-45%, 5%-40%, 10%-40%,15%-40%, 20%-40%, 25%-40%, 30%-40%, 35%-40%, 5%-35%, 10%-35%, 15%-35%,20%-35%, 25%-35%, 30%-35%, 5%-30%, 10%-30%, 15%-30%, 20%-30%, 25%-30%,5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, 15%-20%, 5%-15%,10%-15%, or 5%-10%) of the nucleotides in the double-stranded region ofthe siRNA comprise modified nucleotides.

In further embodiments, the siRNA comprises modified nucleotidesincluding, but not limited to, 2′-O-methyl (2′OMe) nucleotides,2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, and mixtures thereof. In preferred embodiments, the siRNAcomprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, e.g., 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosine nucleotides, ormixtures thereof. In one particular embodiment, the siRNA comprises atleast one 2′OMe-guanosine nucleotide, 2′OMe-uridine nucleotide, ormixtures thereof. In certain instances, the siRNA does not comprise2′OMe-cytosine nucleotides. In other embodiments, the siRNA comprises ahairpin loop structure.

In certain embodiments, the siRNA comprises modified nucleotides in onestrand (i.e., sense or antisense) or both strands of the double-strandedregion of the siRNA molecule. Preferably, uridine and/or guanosinenucleotides are modified at selective positions in the double-strandedregion of the siRNA duplex. With regard to uridine nucleotidemodifications, at least one, two, three, four, five, six, or more of theuridine nucleotides in the sense and/or antisense strand can be amodified uridine nucleotide such as a 2′OMe-uridine nucleotide. In someembodiments, every uridine nucleotide in the sense and/or antisensestrand is a 2′OMe-uridine nucleotide. With regard to guanosinenucleotide modifications, at least one, two, three, four, five, six, ormore of the guanosine nucleotides in the sense and/or antisense strandcan be a modified guanosine nucleotide such as a 2′OMe-guanosinenucleotide. In some embodiments, every guanosine nucleotide in the senseand/or antisense strand is a 2′OMe-guanosine nucleotide.

In certain embodiments, at least one, two, three, four, five, six,seven, or more 5′-GU-3′ motifs in an siRNA sequence may be modified,e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/orby introducing modified nucleotides such as 2′OMe nucleotides. The5′-GU-3′ motif can be in the sense strand, the antisense strand, or bothstrands of the siRNA sequence. The 5′-GU-3′ motifs may be adjacent toeach other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more nucleotides.

In some embodiments, a modified siRNA molecule is less immunostimulatorythan a corresponding unmodified siRNA sequence. In such embodiments, themodified siRNA molecule with reduced immunostimulatory propertiesadvantageously retains RNAi activity against the target sequence. Inanother embodiment, the immunostimulatory properties of the modifiedsiRNA molecule and its ability to silence target gene expression can bebalanced or optimized by the introduction of minimal and selective 2′OMemodifications within the siRNA sequence such as, e.g., within thedouble-stranded region of the siRNA duplex. In certain instances, themodified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that the immunostimulatory properties of the modifiedsiRNA molecule and the corresponding unmodified siRNA molecule can bedetermined by, for example, measuring INF-α and/or IL-6 levels fromabout two to about twelve hours after systemic administration in amammal or transfection of a mammalian responder cell using anappropriate lipid-based delivery system (such as the SNALP deliverysystem disclosed herein).

In other embodiments, a modified siRNA molecule has an IC₅₀ (i.e.,half-maximal inhibitory concentration) less than or equal to ten-foldthat of the corresponding unmodified siRNA (i.e., the modified siRNA hasan IC₅₀ that is less than or equal to ten-times the IC₅₀ of thecorresponding unmodified siRNA). In other embodiments, the modifiedsiRNA has an IC₅₀ less than or equal to three-fold that of thecorresponding unmodified siRNA sequence. In yet other embodiments, themodified siRNA has an IC₅₀ less than or equal to two-fold that of thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that a dose-response curve can be generated and theIC₅₀ values for the modified siRNA and the corresponding unmodifiedsiRNA can be readily determined using methods known to those of skill inthe art.

In another embodiment, an unmodified or modified siRNA molecule iscapable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% of the expression of the target sequencerelative to a negative control (e.g., buffer only, an siRNA sequencethat targets a different gene, a scrambled siRNA sequence, etc.).

In yet another embodiment, a modified siRNA molecule is capable ofsilencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% of the expression of the target sequence relative tothe corresponding unmodified siRNA sequence.

In some embodiments, the siRNA molecule does not comprise phosphatebackbone modifications, e.g., in the sense and/or antisense strand ofthe double-stranded region. In other embodiments, the siRNA comprisesone, two, three, four, or more phosphate backbone modifications, e.g.,in the sense and/or antisense strand of the double-stranded region. Inpreferred embodiments, the siRNA does not comprise phosphate backbonemodifications.

In further embodiments, the siRNA does not comprise 2′-deoxynucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region. In yet further embodiments, the siRNA comprisesone, two, three, four, or more 2′-deoxy nucleotides, e.g., in the senseand/or antisense strand of the double-stranded region. In preferredembodiments, the siRNA does not comprise 2′-deoxy nucleotides.

In certain instances, the nucleotide at the 3′-end of thedouble-stranded region in the sense and/or antisense strand is not amodified nucleotide. In certain other instances, the nucleotides nearthe 3′-end (e.g., within one, two, three, or four nucleotides of the3′-end) of the double-stranded region in the sense and/or antisensestrand are not modified nucleotides.

The siRNA molecules described herein may have 3′ overhangs of one, two,three, four, or more nucleotides on one or both sides of thedouble-stranded region, or may lack overhangs (i.e., have blunt ends) onone or both sides of the double-stranded region. In certain embodiments,the 3′ overhang on the sense and/or antisense strand independentlycomprises one, two, three, four, or more modified nucleotides such as2′OMe nucleotides and/or any other modified nucleotide described hereinor known in the art.

In particular embodiments, siRNAs are administered using a carriersystem such as a nucleic acid-lipid particle. In a preferred embodiment,the nucleic acid-lipid particle comprises: (a) one or more siRNAmolecules targeting APOB, APOC3, PCSK9, DGAT1 and/or DGAT2 geneexpression; (b) a cationic lipid of Formula I-XIV or a salt thereof; and(c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol).In certain instances, the nucleic acid-lipid particle may furthercomprise a conjugated lipid that prevents aggregation of particles(e.g., PEG-DAA).

b) Dicer-Substrate dsRNA

As used herein, the term “Dicer-substrate dsRNA” or “precursor RNAimolecule” is intended to include any precursor molecule that isprocessed in vivo by Dicer to produce an active siRNA which isincorporated into the RISC complex for RNA interference of a targetgene, such as APOB, APOC3, PCSK9, DGAT1, DGAT2, or combinations thereof.

In one embodiment, the Dicer-substrate dsRNA has a length sufficientsuch that it is processed by Dicer to produce an siRNA. According tothis embodiment, the Dicer-substrate dsRNA comprises (i) a firstoligonucleotide sequence (also termed the sense strand) that is betweenabout 25 and about 60 nucleotides in length (e.g., about 25-60, 25-55,25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), preferablybetween about 25 and about 30 nucleotides in length (e.g., 25, 26, 27,28, 29, or 30 nucleotides in length), and (ii) a second oligonucleotidesequence (also termed the antisense strand) that anneals to the firstsequence under biological conditions, such as the conditions found inthe cytoplasm of a cell. The second oligonucleotide sequence may bebetween about 25 and about 60 nucleotides in length (e.g., about 25-60,25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), andis preferably between about 25 and about 30 nucleotides in length (e.g.,25, 26, 27, 28, 29, or 30 nucleotides in length). In addition, a regionof one of the sequences, particularly of the antisense strand, of theDicer-substrate dsRNA has a sequence length of at least about 19nucleotides, for example, from about 19 to about 60 nucleotides (e.g.,about 19-60, 19-55, 19-50, 19-45, 19-40, 19-35, 19-30, or 19-25nucleotides), preferably from about 19 to about 23 nucleotides (e.g.,19, 20, 21, 22, or 23 nucleotides) that are sufficiently complementaryto a nucleotide sequence of the RNA produced from the target gene totrigger an RNAi response.

In a second embodiment, the Dicer-substrate dsRNA has several propertieswhich enhance its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and has at least one of the following properties: (i)the dsRNA is asymmetric, e.g., has a 3′-overhang on the antisensestrand; and/or (ii) the dsRNA has a modified 3′-end on the sense strandto direct orientation of Dicer binding and processing of the dsRNA to anactive siRNA. According to this latter embodiment, the sense strandcomprises from about 22 to about 28 nucleotides and the antisense strandcomprises from about 24 to about 30 nucleotides.

In one embodiment, the Dicer-substrate dsRNA has an overhang on the3′-end of the antisense strand. In another embodiment, the sense strandis modified for Dicer binding and processing by suitable modifierslocated at the 3′-end of the sense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides, and thelike, and sterically hindered molecules such as fluorescent moleculesand the like. When nucleotide modifiers are used, they replaceribonucleotides in the dsRNA such that the length of the dsRNA does notchange. In another embodiment, the Dicer-substrate dsRNA has an overhangon the 3′-end of the antisense strand and the sense strand is modifiedfor Dicer processing. In another embodiment, the 5′-end of the sensestrand has a phosphate. In another embodiment, the 5′-end of theantisense strand has a phosphate. In another embodiment, the antisensestrand or the sense strand or both strands have one or more 2′-O-methyl(2′OMe) modified nucleotides. In another embodiment, the antisensestrand contains 2′OMe modified nucleotides. In another embodiment, theantisense stand contains a 3′-overhang that is comprised of 2′OMemodified nucleotides. The antisense strand could also include additional2′OMe modified nucleotides. The sense and antisense strands anneal underbiological conditions, such as the conditions found in the cytoplasm ofa cell. In addition, a region of one of the sequences, particularly ofthe antisense strand, of the Dicer-substrate dsRNA has a sequence lengthof at least about 19 nucleotides, wherein these nucleotides are in the21-nucleotide region adjacent to the 3′-end of the antisense strand andare sufficiently complementary to a nucleotide sequence of the RNAproduced from the target gene, such as APOB. Further, in accordance withthis embodiment, the Dicer-substrate dsRNA may also have one or more ofthe following additional properties: (a) the antisense strand has aright shift from the typical 21-mer (i.e., the antisense strand includesnucleotides on the right side of the molecule when compared to thetypical 21-mer); (b) the strands may not be completely complementary,i.e., the strands may contain simple mismatch pairings; and (c) basemodifications such as locked nucleic acid(s) may be included in the5′-end of the sense strand.

In a third embodiment, the sense strand comprises from about 25 to about28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides), wherein the 2nucleotides on the 3′-end of the sense strand are deoxyribonucleotides.The sense strand contains a phosphate at the 5′-end. The antisensestrand comprises from about 26 to about 30 nucleotides (e.g., 26, 27,28, 29, or 30 nucleotides) and contains a 3′-overhang of 1-4nucleotides. The nucleotides comprising the 3′-overhang are modifiedwith 2′OMe modified ribonucleotides. The antisense strand containsalternating 2′OMe modified nucleotides beginning at the first monomer ofthe antisense strand adjacent to the 3′-overhang, and extending 15-19nucleotides from the first monomer adjacent to the 3′-overhang. Forexample, for a 27-nucleotide antisense strand and counting the firstbase at the 5′-end of the antisense strand as position number 1, 2′OMemodifications would be placed at bases 9, 11, 13, 15, 17, 19, 21, 23,25, 26, and 27. In one embodiment, the Dicer-substrate dsRNA has thefollowing structure:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′OMe RNA, “Y” is anoverhang domain comprised of 1, 2, 3, or 4 RNA monomers that areoptionally 2′OMe RNA monomers, and “D”=DNA. The top strand is the sensestrand, and the bottom strand is the antisense strand.

In a fourth embodiment, the Dicer-substrate dsRNA has several propertieswhich enhance its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and at least one of the following properties: (i) thedsRNA is asymmetric, e.g., has a 3′-overhang on the sense strand; and(ii) the dsRNA has a modified 3′-end on the antisense strand to directorientation of Dicer binding and processing of the dsRNA to an activesiRNA. According to this embodiment, the sense strand comprises fromabout 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30nucleotides) and the antisense strand comprises from about 22 to about28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides). In oneembodiment, the Dicer-substrate dsRNA has an overhang on the 3′-end ofthe sense strand. In another embodiment, the antisense strand ismodified for Dicer binding and processing by suitable modifiers locatedat the 3′-end of the antisense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides, and thelike, and sterically hindered molecules such as fluorescent moleculesand the like. When nucleotide modifiers are used, they replaceribonucleotides in the dsRNA such that the length of the dsRNA does notchange. In another embodiment, the dsRNA has an overhang on the 3′-endof the sense strand and the antisense strand is modified for Dicerprocessing. In one embodiment, the antisense strand has a 5′-phosphate.The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are adjacent to the 3′-end of antisense strand and aresufficiently complementary to a nucleotide sequence of the RNA producedfrom the target gene, such as APOB. Further, in accordance with thisembodiment, the Dicer-substrate dsRNA may also have one or more of thefollowing additional properties: (a) the antisense strand has a leftshift from the typical 21-mer (i.e., the antisense strand includesnucleotides on the left side of the molecule when compared to thetypical 21-mer); and (b) the strands may not be completelycomplementary, i.e., the strands may contain simple mismatch pairings.

In a preferred embodiment, the Dicer-substrate dsRNA has an asymmetricstructure, with the sense strand having a 25-base pair length, and theantisense strand having a 27-base pair length with a 2 base 3′-overhang.In certain instances, this dsRNA having an asymmetric structure furthercontains 2 deoxynucleotides at the 3′-end of the sense strand in placeof two of the ribonucleotides. In certain other instances, this dsRNAhaving an asymmetric structure further contains 2′OMe modifications atpositions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense strand(wherein the first base at the 5′-end of the antisense strand isposition 1). In certain additional instances, this dsRNA having anasymmetric structure further contains a 3′-overhang on the antisensestrand comprising 1, 2, 3, or 4 2′OMe nucleotides (e.g., a 3′-overhangof 2′OMe nucleotides at positions 26 and 27 on the antisense strand).

In another embodiment, Dicer-substrate dsRNAs may be designed by firstselecting an antisense strand siRNA sequence having a length of at least19 nucleotides. In some instances, the antisense siRNA is modified toinclude about 5 to about 11 ribonucleotides on the 5′-end to provide alength of about 24 to about 30 nucleotides. When the antisense strandhas a length of 21 nucleotides, 3-9, preferably 4-7, or more preferably6 nucleotides may be added on the 5′-end. Although the addedribonucleotides may be complementary to the target gene sequence, fullcomplementarity between the target sequence and the antisense siRNA isnot required. That is, the resultant antisense siRNA is sufficientlycomplementary with the target sequence. A sense strand is then producedthat has about 22 to about 28 nucleotides. The sense strand issubstantially complementary with the antisense strand to anneal to theantisense strand under biological conditions. In one embodiment, thesense strand is synthesized to contain a modified 3′-end to direct Dicerprocessing of the antisense strand. In another embodiment, the antisensestrand of the dsRNA has a 3′-overhang. In a further embodiment, thesense strand is synthesized to contain a modified 3′-end for Dicerbinding and processing and the antisense strand of the dsRNA has a3′-overhang.

In a related embodiment, the antisense siRNA may be modified to includeabout 1 to about 9 ribonucleotides on the 5′-end to provide a length ofabout 22 to about 28 nucleotides. When the antisense strand has a lengthof 21 nucleotides, 1-7, preferably 2-5, or more preferably 4ribonucleotides may be added on the 3′-end. The added ribonucleotidesmay have any sequence. Although the added ribonucleotides may becomplementary to the target gene sequence, full complementarity betweenthe target sequence, and the antisense siRNA is not required. That is,the resultant antisense siRNA is sufficiently complementary with thetarget sequence. A sense strand is then produced that has about 24 toabout 30 nucleotides. The sense strand is substantially complementarywith the antisense strand to anneal to the antisense strand underbiological conditions. In one embodiment, the antisense strand issynthesized to contain a modified 3′-end to direct Dicer processing. Inanother embodiment, the sense strand of the dsRNA has a 3′-overhang. Ina further embodiment, the antisense strand is synthesized to contain amodified 3′-end for Dicer binding and processing and the sense strand ofthe dsRNA has a 3′-overhang.

Suitable Dicer-substrate dsRNA sequences can be identified, synthesized,and modified using any means known in the art for designing,synthesizing, and modifying siRNA sequences. In particular embodiments,Dicer-substrate dsRNAs are administered using a carrier system such as anucleic acid-lipid particle. In a preferred embodiment, the nucleicacid-lipid particle comprises: (a) one or more Dicer-substrate dsRNAmolecules targeting APOB, APOC3, PCSK9, DGAT1 and/or DGAT2 geneexpression; (b) a cationic lipid of Formula I-XIV or a salt thereof; and(c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol).In certain instances, the nucleic acid-lipid particle may furthercomprise a conjugated lipid that prevents aggregation of particles(e.g., PEG-DAA).

Additional embodiments related to the Dicer-substrate dsRNAs of theinvention, as well as methods of designing and synthesizing such dsRNAs,are described in U.S. Patent Publication Nos. 20050244858, 20050277610,and 20070265220, and U.S. application Ser. No. 12/794,701, filed Jun. 4,2010, the disclosures of which are herein incorporated by reference intheir entirety for all purposes.

c) Small Hairpin RNA (shRNA)

A “small hairpin RNA” or “short hairpin RNA” or “shRNA” includes a shortRNA sequence that makes a tight hairpin turn that can be used to silencegene expression via RNA interference. The shRNAs of the invention may bechemically synthesized or transcribed from a transcriptional cassette ina DNA plasmid. The shRNA hairpin structure is cleaved by the cellularmachinery into siRNA, which is then bound to the RNA-induced silencingcomplex (RISC).

The shRNAs of the invention are typically about 15-60, 15-50, or 15-40(duplex) nucleotides in length, more typically about 15-30, 15-25, or19-25 (duplex) nucleotides in length, and are preferably about 20-24,21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementarysequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30,15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or21-23 nucleotides in length, and the double-stranded shRNA is about15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length,preferably about 18-22, 19-20, or 19-21 base pairs in length). shRNAduplexes may comprise 3′ overhangs of about 1 to about 4 nucleotides orabout 2 to about 3 nucleotides on the antisense strand and/or5′-phosphate termini on the sense strand. In some embodiments, the shRNAcomprises a sense strand and/or antisense strand sequence of from about15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50,15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), preferablyfrom about 19 to about 40 nucleotides in length (e.g., about 19-40,19-35, 19-30, or 19-25 nucleotides in length), more preferably fromabout 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23nucleotides in length).

Non-limiting examples of shRNA include a double-stranded polynucleotidemolecule assembled from a single-stranded molecule, where the sense andantisense regions are linked by a nucleic acid-based or non-nucleicacid-based linker; and a double-stranded polynucleotide molecule with ahairpin secondary structure having self-complementary sense andantisense regions. In preferred embodiments, the sense and antisensestrands of the shRNA are linked by a loop structure comprising fromabout 1 to about 25 nucleotides, from about 2 to about 20 nucleotides,from about 4 to about 15 nucleotides, from about 5 to about 12nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.

Additional shRNA sequences include, but are not limited to, asymmetricshRNA precursor polynucleotides such as those described in PCTPublication Nos. WO 2006/074108 and WO 2009/076321, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. For example, PCT Publication No. WO 2006/074108 disclosesself-protected oligonucleotides comprising a region having a sequencecomplementary to one, two, three, or more same or different target mRNAsequences (e.g., multivalent shRNAs) and one or more self-complementaryregions. Similarly, PCT Publication No. WO 2009/076321 disclosesself-forming asymmetric precursor polynucleotides comprising a targetingregion comprising a polynucleotide sequence complementary to a region ofone, two, three, or more same or different target mRNA sequences (e.g.,multivalent shRNAs); a first self-complementary region; and a secondself-complementary region, wherein the first and secondself-complementary regions are located one at each end of the targetingregion and both self-complementary regions form stem-loop structures,wherein the first self-complementary region is capable of being cleavedby a RNase III endoribonuclease that is not a class IV DICERendoribonuclease, and wherein both self-complementary regions comprise anucleotide sequence that is complementary to a region of the target genesequence, but wherein a portion of the target sequence present in thetargeting region does not have a complementary sequence in either of theself-complementary regions.

Suitable shRNA sequences can be identified, synthesized, and modifiedusing any means known in the art for designing, synthesizing, andmodifying siRNA sequences. In particular embodiments, shRNAs areadministered using a carrier system such as a nucleic acid-lipidparticle. In a preferred embodiment, the nucleic acid-lipid particlecomprises: (a) one or more shRNA molecules targeting APOB, APOC3, PCSK9,DGAT1 and/or DGAT2 gene expression; (b) a cationic lipid of FormulaI-XIV or a salt thereof; and (c) a non-cationic lipid (e.g., DPPC, DSPC,DSPE, and/or cholesterol). In certain instances, the nucleic acid-lipidparticle may further comprise a conjugated lipid that preventsaggregation of particles (e.g., PEG-DAA).

Additional embodiments related to the shRNAs of the invention, as wellas methods of designing and synthesizing such shRNAs, are described inU.S. application Ser. No. 12/794,701, filed Jun. 4, 2010, the disclosureof which is herein incorporated by reference in its entirety for allpurposes.

d) aiRNA

Like siRNA, asymmetrical interfering RNA (aiRNA) can recruit theRNA-induced silencing complex (RISC) and lead to effective silencing ofa variety of genes in mammalian cells by mediating sequence-specificcleavage of the target sequence between nucleotide 10 and 11 relative tothe 5′ end of the antisense strand (Sun et al., Nat. Biotech.,26:1379-1382 (2008)). Typically, an aiRNA molecule comprises a short RNAduplex having a sense strand and an antisense strand, wherein the duplexcontains overhangs at the 3′ and 5′ ends of the antisense strand. TheaiRNA is generally asymmetric because the sense strand is shorter onboth ends when compared to the complementary antisense strand. In someaspects, aiRNA molecules may be designed, synthesized, and annealedunder conditions similar to those used for siRNA molecules. As anon-limiting example, aiRNA sequences may be selected and generatedusing the methods described above for selecting siRNA sequences.

In another embodiment, aiRNA duplexes of various lengths (e.g., about10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more typically12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs) may be designed withoverhangs at the 3′ and 5′ ends of the antisense strand to target anmRNA of interest. In certain instances, the sense strand of the aiRNAmolecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or20 nucleotides in length. In certain other instances, the antisensestrand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and is preferably about 20-24, 21-22, or 21-23 nucleotides inlength.

In some embodiments, the 5′ antisense overhang contains one, two, three,four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”, etc.).In other embodiments, the 3′ antisense overhang contains one, two,three, four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”,etc.). In certain aspects, the aiRNA molecules described herein maycomprise one or more modified nucleotides, e.g., in the double-stranded(duplex) region and/or in the antisense overhangs. As a non-limitingexample, aiRNA sequences may comprise one or more of the modifiednucleotides described above for siRNA sequences. In a preferredembodiment, the aiRNA molecule comprises 2′OMe nucleotides such as, forexample, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, ormixtures thereof.

In certain embodiments, aiRNA molecules may comprise an antisense strandwhich corresponds to the antisense strand of an siRNA molecule, e.g.,one of the siRNA molecules described herein. In particular embodiments,aiRNAs are administered using a carrier system such as a nucleicacid-lipid particle. In a preferred embodiment, the nucleic acid-lipidparticle comprises: (a) one or more aiRNA molecules targeting APOB,APOC3, PCSK9, DGAT1 and/or DGAT2 gene expression; (b) a cationic lipidof Formula I-XIV or a salt thereof; and (c) a non-cationic lipid (e.g.,DPPC, DSPC, DSPE, and/or cholesterol). In certain instances, the nucleicacid-lipid particle may further comprise a conjugated lipid thatprevents aggregation of particles (e.g., PEG-DAA).

Suitable aiRNA sequences can be identified, synthesized, and modifiedusing any means known in the art for designing, synthesizing, andmodifying siRNA sequences. Additional embodiments related to the aiRNAmolecules of the invention are described in U.S. Patent Publication No.20090291131 and PCT Publication No. WO 09/127,060, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

e) miRNA

Generally, microRNAs (miRNA) are single-stranded RNA molecules of about21-23 nucleotides in length which regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed, but miRNAs are nottranslated into protein (non-coding RNA); instead, each primarytranscript (a pri-miRNA) is processed into a short stem-loop structurecalled a pre-miRNA and finally into a functional mature miRNA. MaturemiRNA molecules are either partially or completely complementary to oneor more messenger RNA (mRNA) molecules, and their main function is todownregulate gene expression. The identification of miRNA molecules isdescribed, e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau etal., Science, 294:858-862; and Lee et al., Science, 294:862-864.

The genes encoding miRNA are much longer than the processed mature miRNAmolecule. miRNA are first transcribed as primary transcripts orpri-miRNA with a cap and poly-A tail and processed to short,˜70-nucleotide stem-loop structures known as pre-miRNA in the cellnucleus. This processing is performed in animals by a protein complexknown as the Microprocessor complex, consisting of the nuclease Droshaand the double-stranded RNA binding protein Pasha (Denli et al., Nature,432:231-235 (2004)). These pre-miRNA are then processed to mature miRNAin the cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC)(Bernstein et al., Nature, 409:363-366 (2001). Either the sense strandor antisense strand of DNA can function as templates to give rise tomiRNA.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC complex.This strand is known as the guide strand and is selected by theargonaute protein, the catalytically active RNase in the RISC complex,on the basis of the stability of the 5′ end (Preall et al., Curr. Biol.,16:530-535 (2006)). The remaining strand, known as the anti-guide orpassenger strand, is degraded as a RISC complex substrate (Gregory etal., Cell, 123:631-640 (2005)). After integration into the active RISCcomplex, miRNAs base pair with their complementary mRNA molecules andinduce target mRNA degradation and/or translational silencing.

Mammalian miRNA molecules are usually complementary to a site in the 3′UTR of the target mRNA sequence. In certain instances, the annealing ofthe miRNA to the target mRNA inhibits protein translation by blockingthe protein translation machinery. In certain other instances, theannealing of the miRNA to the target mRNA facilitates the cleavage anddegradation of the target mRNA through a process similar to RNAinterference (RNAi). miRNA may also target methylation of genomic siteswhich correspond to targeted mRNA. Generally, miRNA function inassociation with a complement of proteins collectively termed the miRNP.

In certain aspects, the miRNA molecules described herein are about15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and are preferably about 20-24, 21-22, or 21-23 nucleotides inlength. In certain other aspects, miRNA molecules may comprise one ormore modified nucleotides. As a non-limiting example, miRNA sequencesmay comprise one or more of the modified nucleotides described above forsiRNA sequences. In a preferred embodiment, the miRNA molecule comprises2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, or mixtures thereof.

In particular embodiments, miRNAs are administered using a carriersystem such as a nucleic acid-lipid particle. In a preferred embodiment,the nucleic acid-lipid particle comprises: (a) one or more miRNAmolecules targeting APOB, APOC3, PCSK9, DGAT1 and/or DGAT2 geneexpression; (b) a cationic lipid of Formula I-XIV or a salt thereof; and(c) a non-cationic lipid (e.g., DPPC, DSPC, DSPE, and/or cholesterol).In certain instances, the nucleic acid-lipid particle may furthercomprise a conjugated lipid that prevents aggregation of particles(e.g., PEG-DAA).

In other embodiments, one or more agents that block the activity of anmiRNA targeting APOB, APOC3, PCSK9, DGAT1 and/or DGAT2 mRNA areadministered using a lipid particle of the invention (e.g., a nucleicacid-lipid particle such as SNALP). Examples of blocking agents include,but are not limited to, steric blocking oligonucleotides, locked nucleicacid oligonucleotides, and Morpholino oligonucleotides. Such blockingagents may bind directly to the miRNA or to the miRNA binding site onthe target mRNA.

Additional embodiments related to the miRNA molecules of the inventionare described in U.S. Patent Publication No. 20090291131 and PCTPublication No. WO 09/127,060, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

B. Cationic Lipids

Any of the cationic lipids of Formulas I-XIV or salts thereof as setforth herein may be used in the lipid particles of the present invention(e.g., SNALP), either alone or in combination with one or more othercationic lipid species or non-cationic lipid species. The cationiclipids include the (R) and/or (S) enantiomers thereof.

In some embodiments, the cationic lipid comprises a racemic mixture. Inother embodiments, the cationic lipid comprises a mixture of one or morediastereomers. In certain embodiments, the cationic lipid is enriched inone enantiomer, such that the cationic lipid comprises at least about55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% enantiomeric excess. Incertain other embodiments, the cationic lipid is enriched in onediastereomer, such that the cationic lipid comprises at least about 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% diastereomeric excess. Incertain additional embodiments, the cationic lipid is chirally pure(e.g., comprises a single optical isomer). In further embodiments, thecationic lipid is enriched in one optical isomer (e.g., an opticallyactive isomer), such that the cationic lipid comprises at least about55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% isomeric excess. Thepresent invention provides the synthesis of the cationic lipids ofFormulas I-XIV as a racemic mixture or in optically pure form.

The terms “cationic lipid” and “amino lipid” are used interchangeablyherein to include those lipids and salts thereof having one, two, three,or more fatty acid or fatty alkyl chains and a pH-titratable amino headgroup (e.g., an alkylamino or dialkylamino head group). The cationiclipid is typically protonated (i.e., positively charged) at a pH belowthe pK_(a) of the cationic lipid and is substantially neutral at a pHabove the pK_(a). The cationic lipids of the invention may also betermed titratable cationic lipids.

The term “salts” includes any anionic and cationic complex, such as thecomplex formed between a cationic lipid disclosed herein and one or moreanions. Non-limiting examples of anions include inorganic and organicanions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate(e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate,dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite,nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate,hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate,lactate, acrylate, polyacrylate, fumarate, maleate, itaconate,glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate,polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite,bromate, hypobromite, iodate, an alkylsulfonate, an arylsulfonate,arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate,hydroxide, peroxide, permanganate, and mixtures thereof. In particularembodiments, the salts of the cationic lipids disclosed herein arecrystalline salts.

The term “alkyl” includes a straight chain or branched, noncyclic orcyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbonatoms. Representative saturated straight chain alkyls include, but arenot limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, andthe like, while saturated branched alkyls include, without limitation,isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Representative saturated cyclic alkyls include, but are not limited to,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like, whileunsaturated cyclic alkyls include, without limitation, cyclopentenyl,cyclohexenyl, and the like.

The term “alkenyl” includes an alkyl, as defined above, containing atleast one double bond between adjacent carbon atoms. Alkenyls includeboth cis and trans isomers. Representative straight chain and branchedalkenyls include, but are not limited to, ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike.

The term “alkynyl” includes any alkyl or alkenyl, as defined above,which additionally contains at least one triple bond between adjacentcarbons. Representative straight chain and branched alkynyls include,without limitation, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

The term “acyl” includes any alkyl, alkenyl, or alkynyl wherein thecarbon at the point of attachment is substituted with an oxo group, asdefined below. The following are non-limiting examples of acyl groups:—C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl.

The term “heterocycle” includes a 5- to 7-membered monocyclic, or 7- to10-membered bicyclic, heterocyclic ring which is either saturated,unsaturated, or aromatic, and which contains from 1 or 2 heteroatomsindependently selected from nitrogen, oxygen and sulfur, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen heteroatom may be optionally quaternized, including bicyclicrings in which any of the above heterocycles are fused to a benzenering. The heterocycle may be attached via any heteroatom or carbon atom.Heterocycles include, but are not limited to, heteroaryls as definedbelow, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl,oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” mean that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O), two hydrogen atoms are replaced.In this regard, substituents include, but are not limited to, oxo,halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y),—NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y),—SO_(n)R^(x), and —SO_(n)NR^(x)R^(y), wherein n is 0, 1, or 2, R^(x) andR^(y) are the same or different and are independently hydrogen, alkyl,or heterocycle, and each of the alkyl and heterocycle substituents maybe further substituted with one or more of oxo, halogen, —OH, —CN,alkyl, —OR^(x), heterocycle, —NR^(x)R^(y), —NR^(x)C(═O)R^(y),—NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y),—SO_(n)R^(x), and —SO_(n)NR^(x)R^(y). The term “optionally substituted,”when used before a list of substituents, means that each of thesubstituents in the list may be optionally substituted as describedherein.

The term “halogen” includes fluoro, chloro, bromo, and iodo.

In one aspect, cationic lipids of Formula I having the followingstructure (or salts thereof) are useful in the present invention:

wherein R¹ and R² are either the same or different and are independentlyan optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄alkynyl, or C₁₂-C₂₄ acyl; R³ and R⁴ are either the same or different andare independently an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,or C₂-C₆ alkynyl, or R³ and R⁴ may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms chosen from nitrogen and oxygen; R⁵ is either absent or ishydrogen (H) or a C₁-C₆ alkyl to provide a quaternary amine; m, n, and pare either the same or different and are independently either 0, 1, or2, with the proviso that m, n, and p are not simultaneously 0; q is 0,1, 2, 3, or 4; and Y and Z are either the same or different and areindependently O, S, or NH.

In some embodiments, R³ and R⁴ are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R³ and R⁴ are both methyl groups. In one embodiment, q is 1or 2. In another embodiment, q is 1-2, 1-3, 1-4, 2-3, or 2-4. In furtherembodiments, R⁵ is absent when the pH is above the pK_(a) of thecationic lipid and R⁵ is hydrogen when the pH is below the pK_(a) of thecationic lipid such that the amino head group is protonated. In analternative embodiment, R⁵ is an optionally substituted C₁-C₄ alkyl toprovide a quaternary amine. In additional embodiments, Y and Z are bothO.

In other embodiments, R¹ and R² are independently an optionallysubstituted C₁₂-C₂₄, C₁₂-C₂₂, C₁₂-C₂₀, C₁₄-C₂₄, C₁₄-C₂₂, C₁₄-C₂₀,C₁₆-C₂₄, C₁₆-C₂₂, or C₁₆-C₂₀ alkyl, alkenyl, alkynyl, or acyl group(i.e., C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, orC₂₄ alkyl, alkenyl, alkynyl, or acyl group). In certain embodiments, atleast one or both R¹ and R² independently comprises at least 1, 2, 3, 4,5, or 6 sites of unsaturation (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4,2-5, or 2-6 sites of unsaturation) or a substituted alkyl or acyl group.In certain instances, the unsaturated side-chain may comprise amyristoleyl moiety, a palmitoleyl moiety, an oleyl moiety, adodecadienyl moiety, a tetradecadienyl moiety, a hexadecadienyl moiety,an octadecadienyl moiety, an icosadienyl moiety, a dodecatrienyl moiety,a tetradectrienyl moiety, a hexadecatrienyl moiety, an octadecatrienylmoiety, an icosatrienyl moiety, or an acyl derivative thereof (e.g.,linoleoyl, linolenoyl, γ-linolenoyl, etc.). In some instances, theoctadecadienyl moiety is a linoleyl moiety. In particular embodiments,R¹ and R² are both linoleyl moieties. In other instances, theoctadecatrienyl moiety is a linolenyl moiety or a γ-linolenyl moiety. Inparticular embodiments, R¹ and R² are both linolenyl moieties orγ-linolenyl moieties.

In embodiments where one or both R¹ and R² independently comprises atleast 1, 2, 3, 4, 5, or 6 sites of unsaturation, the double bondspresent in one or both R¹ and R² may be in the cis and/or transconfiguration. In certain instances, R¹ and R² are both the same, e.g.,R¹ and R² are both linoleyl (C₁₈) moieties, etc. In certain otherinstances, R¹ and R² are different, e.g., R¹ is a tetradectrienyl (C₁₄)moiety and R² is a linoleyl (C₁₈) moiety. In a preferred embodiment, thecationic lipid of Formula I is symmetrical, i.e., R¹ and R² are both thesame. In another preferred embodiment, at least one or both R¹ and R²comprises at least two sites of unsaturation (e.g., 2, 3, 4, 5, 6, 2-3,2-4, 2-5, or 2-6 sites of unsaturation).

In embodiments where one or both R¹ and R² independently comprises abranched alkyl or acyl group (e.g., a substituted alkyl or acyl group),the branched alkyl or acyl group may comprise a C₁₂-C₂₄ alkyl or acylhaving at least 1-6 (e.g., 1, 2, 3, 4, 5, 6, or more) C₁-C₆ alkylsubstituents. In particular embodiments, the branched alkyl or acylgroup comprises a C₁₂-C₂₀ or C₁₄-C₂₂ alkyl or acyl with 1-6 (e.g., 1, 2,3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl, ethyl, propyl, or butyl)substituents. In some embodiments, the branched alkyl group comprises aphytanyl (3,7,11,15-tetramethyl-hexadecanyl) moiety and the branchedacyl group comprises a phytanoyl (3,7,11,15-tetramethyl-hexadecanoyl)moiety. In particular embodiments, R¹ and R² are both phytanyl moieties.

In some groups of embodiments to the cationic lipids of Formula I, R¹and R² are either the same or different and are independently selectedfrom the group consisting of:

In certain embodiments, cationic lipids falling within the scope ofFormula I include, but are not limited to, the following:2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2” or “C2K”), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)[1,3]-dioxolane(DLin-K-C3-DMA; “C3K”),2,2-dilinoleyl-4-(4-dimethylaminobutyl)[1,3]-dioxolane (DLin-K-C4-DMA;“C4K”), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane(DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane(DLin-K-MPZ), 2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane(DO-K-DMA), 2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane(DS-K-DMA), 2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane (DLin-K-MA),2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride(DLin-K-TMA.Cl),2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane(DLin-K²-DMA), 2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane(D-Lin-K-N-methylpiperzine), DLen-C2K-DMA, γ-DLen-C2K-DMA, DPan-C2K-DMA,DPan-C3K-DMA, or mixtures thereof. In preferred embodiments, thecationic lipid of Formula I comprises DLin-K-C2-DMA and/or DLin-K-DMA.

In some embodiments, the cationic lipids of Formula I form a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula I is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of cationic lipids such as DLin-K-C2-DMA, DLin-K-C3-DMA,DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ, DO-K-DMA, DS-K-DMA, DLin-K-MA,DLin-K-TMA.Cl, DLin-K²-DMA, D-Lin-K-N-methylpiperzine, as well asadditional cationic lipids, is described in PCT Publication No. WO2010/042877, the disclosure of which is incorporated herein by referencein its entirety for all purposes.

The synthesis of cationic lipids such as DLin-K-DMA, as well asadditional cationic lipids, is described in PCT Publication No. WO09/086,558, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

In a preferred embodiment, cationic lipids of Formula II having thefollowing structure (or salts thereof) are useful in the presentinvention:

wherein R¹ and R² are either the same or different and are independentlyan optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄alkynyl, or C₁₂-C₂₄ acyl; R³ and R⁴ are either the same or different andare independently an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl,or C₂-C₆ alkynyl, or R³ and R⁴ may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms chosen from nitrogen and oxygen; R⁵ is either absent or ishydrogen (H) or a C₁-C₆ alkyl to provide a quaternary amine; m, n, and pare either the same or different and are independently either 0, 1, or2, with the proviso that m, n, and p are not simultaneously 0; and Y andZ are either the same or different and are independently O, S, or NH.

In some embodiments, R³ and R⁴ are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R³ and R⁴ are both methyl groups. In further embodiments, R⁵is absent when the pH is above the pK_(a) of the cationic lipid and R⁵is hydrogen when the pH is below the pK_(a) of the cationic lipid suchthat the amino head group is protonated. In an alternative embodiment,R⁵ is an optionally substituted C₁-C₄ alkyl to provide a quaternaryamine. In additional embodiments, Y and Z are both O.

In other embodiments, R¹ and R² are independently an optionallysubstituted C₁₂-C₂₄, C₁₂-C₂₂, C₁₂-C₂₀, C₁₄-C₂₄, C₁₄-C₂₂, C₁₄-C₂₀,C₁₆-C₂₄, C₁₆-C₂₂, or C₁₆-C₂₀ alkyl, alkenyl, alkynyl, or acyl group(i.e., C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, orC₂₄ alkyl, alkenyl, alkynyl, or acyl group). In certain embodiments, atleast one or both R¹ and R² independently comprises at least 1, 2, 3, 4,5, or 6 sites of unsaturation (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4,2-5, or 2-6 sites of unsaturation) or a substituted alkyl or acyl group.In certain instances, the unsaturated side-chain may comprise amyristoleyl moiety, a palmitoleyl moiety, an oleyl moiety, adodecadienyl moiety, a tetradecadienyl moiety, a hexadecadienyl moiety,an octadecadienyl moiety, an icosadienyl moiety, a dodecatrienyl moiety,a tetradectrienyl moiety, a hexadecatrienyl moiety, an octadecatrienylmoiety, an icosatrienyl moiety, or an acyl derivative thereof (e.g.,linoleoyl, linolenoyl, γ-linolenoyl, etc.). In some instances, theoctadecadienyl moiety is a linoleyl moiety. In particular embodiments,R¹ and R² are both linoleyl moieties. In other instances, theoctadecatrienyl moiety is a linolenyl moiety or a γ-linolenyl moiety. Inparticular embodiments, R¹ and R² are both linolenyl moieties orγ-linolenyl moieties.

In embodiments where one or both R¹ and R² independently comprises atleast 1, 2, 3, 4, 5, or 6 sites of unsaturation, the double bondspresent in one or both R¹ and R² may be in the cis and/or transconfiguration. In certain instances, R¹ and R² are both the same, e.g.,R¹ and R² are both linoleyl (C₁₈) moieties, etc. In certain otherinstances, R¹ and R² are different, e.g., R¹ is a tetradectrienyl (C₁₄)moiety and R² is a linoleyl (C₁₈) moiety. In a preferred embodiment, thecationic lipid of Formula II is symmetrical, i.e., R¹ and R² are boththe same. In another preferred embodiment, at least one or both R¹ andR² comprises at least two sites of unsaturation (e.g., 2, 3, 4, 5, 6,2-3, 2-4, 2-5, or 2-6 sites of unsaturation).

In embodiments where one or both R¹ and R² independently comprises abranched alkyl or acyl group (e.g., a substituted alkyl or acyl group),the branched alkyl or acyl group may comprise a C₁₂-C₂₄ alkyl or acylhaving at least 1-6 (e.g., 1, 2, 3, 4, 5, 6, or more) C₁-C₆ alkylsubstituents. In particular embodiments, the branched alkyl or acylgroup comprises a C₁₂-C₂₀ or C₁₄-C₂₂ alkyl or acyl with 1-6 (e.g., 1, 2,3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl, ethyl, propyl, or butyl)substituents. In some embodiments, the branched alkyl group comprises aphytanyl (3,7,11,15-tetramethyl-hexadecanyl) moiety and the branchedacyl group comprises a phytanoyl (3,7,11,15-tetramethyl-hexadecanoyl)moiety. In particular embodiments, R¹ and R² are both phytanyl moieties.

In some groups of embodiments to the cationic lipids of Formula II, R¹and R² are either the same or different and are independently selectedfrom the group consisting of:

In certain embodiments, cationic lipids falling within the scope ofFormula II include, but are not limited to, the following:2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2” or “C2K”), DLen-C2K-DMA, γ-DLen-C2K-DMA, DPan-C2K-DMA, ormixtures thereof. In preferred embodiments, the cationic lipid ofFormula II comprises DLin-K-C2-DMA.

In some embodiments, the cationic lipids of Formula II form a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula II is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of DLin-K-C2-DMA is described herein and in PCTPublication No. WO 2010/042877, the disclosure of which is incorporatedherein by reference in its entirety for all purposes.

In a further aspect, cationic lipids of Formula III having the followingstructure are useful in the present invention:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either absentor present and when present are either the same or different and areindependently an optionally substituted C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl;and n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, R⁴ and R⁵ are both butyl groups. In yet another preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substituted C₂-C₆or C₂-C₄ alkyl or C₂-C₆ or C₂-C₄ alkenyl.

In an alternative embodiment, the cationic lipid of Formula IIIcomprises ester linkages between the amino head group and one or both ofthe alkyl chains. In some embodiments, the cationic lipid of Formula IIIforms a salt (preferably a crystalline salt) with one or more anions. Inone particular embodiment, the cationic lipid of Formula III is theoxalate (e.g., hemioxalate) salt thereof, which is preferably acrystalline salt.

Although each of the alkyl chains in Formula III contains cis doublebonds at positions 6, 9, and 12 (i.e., cis,cis,cis-Δ⁶,Δ⁹,Δ¹²), in analternative embodiment, one, two, or three of these double bonds in oneor both alkyl chains may be in the trans configuration.

In a particularly preferred embodiment, the cationic lipid of FormulaIII has the structure:

In another aspect, cationic lipids of Formula IV having the followingstructure are useful in the present invention:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either the sameor different and are independently an optionally substituted C₁₂-C₂₄alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, wherein atleast one of R⁴ and R⁵ comprises at least three sites of unsaturation ora substituted C₁₂-C₂₄ alkyl; m, n, and p are either the same ordifferent and are independently either 0, 1, or 2, with the proviso thatm, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Zare either the same or different and are independently O, S, or NH.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, q is 2. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In embodiments where at least one of R⁴ and R⁵ comprises a branchedalkyl group (e.g., a substituted C₁₂-C₂₄ alkyl group), the branchedalkyl group may comprise a C₁₂-C₂₄ alkyl having at least 1-6 (e.g., 1,2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. In particularembodiments, the branched alkyl group comprises a C₁₂-C₂₀ or C₁₄-C₂₂alkyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl,ethyl, propyl, or butyl) substituents. Preferably, the branched alkylgroup comprises a phytanyl (3,7,11,15-tetramethyl-hexadecanyl) moiety.In other preferred embodiments, R⁴ and R⁵ are both phytanyl moieties.

In alternative embodiments, at least one of R⁴ and R⁵ comprises abranched acyl group (e.g., a substituted C₁₂-C₂₄ acyl group). In certaininstances, the branched acyl group may comprise a C₁₂-C₂₄ acyl having atleast 1-6 (e.g., 1, 2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. Inparticular embodiments, the branched acyl group comprises a C₁₂-C₂₀ orC₁₄-C₂₂ acyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g.,methyl, ethyl, propyl, or butyl) substituents. Preferably, the branchedacyl group comprises a phytanoyl (3,7,11,15-tetramethyl-hexadecanoyl)moiety.

In embodiments where at least one of R⁴ and R⁵ comprises at least threesites of unsaturation, the double bonds present in one or both alkylchains may be in the cis and/or trans configuration. In someembodiments, R⁴ and R⁵ are independently selected from the groupconsisting of a dodecatrienyl moiety, a tetradectrienyl moiety, ahexadecatrienyl moiety, an octadecatrienyl moiety, an icosatrienylmoiety, and a phytanyl moiety, as well as acyl derivatives thereof(e.g., linolenoyl, γ-linolenoyl, phytanoyl, etc.). In certain instances,the octadecatrienyl moiety is a linolenyl moiety or a γ-linolenylmoiety. In preferred embodiments, R⁴ and R⁵ are both linolenyl moietiesor γ-linolenyl moieties. In particular embodiments, R⁴ and R⁵independently comprise a backbone of from about 16 to about 22 carbonatoms, and one or both of R⁴ and R⁵ independently comprise at leastthree, four, five, or six sites of unsaturation.

In some embodiments, the cationic lipid of Formula IV forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula IV is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula IVhas a structure selected from the group consisting of:

In yet another aspect, cationic lipids of Formula V having the followingstructure are useful in the present invention:

or salts thereof, wherein: R¹ and R² are joined to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either the sameor different and are independently an optionally substituted C₁₂-C₂₄alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and n is 0, 1,2, 3, or 4.

In some embodiments, R¹ and R² are joined to form a heterocyclic ring of5 carbon atoms and 1 nitrogen atom. In certain instances, theheterocyclic ring is substituted with a substituent such as a hydroxylgroup at the ortho, meta, and/or para positions. In a preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In certain embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, and abranched alkyl group as described above (e.g., a phytanyl moiety), aswell as acyl derivatives thereof (e.g., linoleoyl, linolenoyl,γ-linolenoyl, phytanoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In particularembodiments, R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties,γ-linolenyl moieties, or phytanyl moieties.

In some embodiments, the cationic lipid of Formula V forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula V is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula Vhas a structure selected from the group consisting of:

In still yet another aspect, cationic lipids of Formula VI having thefollowing structure are useful in the present invention:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either the sameor different and are independently an optionally substituted C₁₂-C₂₄alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and n is 2, 3,or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, n is 2. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In certain embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, and abranched alkyl group as described above (e.g., a phytanyl moiety), aswell as acyl derivatives thereof (e.g., linoleoyl, linolenoyl,γ-linolenoyl, phytanoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In particularembodiments, R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties,γ-linolenyl moieties, or phytanyl moieties.

In some embodiments, the cationic lipid of Formula VI forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula VI is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula VIhas a structure selected from the group consisting of:

In another aspect, cationic lipids of Formula VII having the followingstructure are useful in the present invention:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are different andare independently an optionally substituted C₁-C₂₄ alkyl, C₂-C₂₄alkenyl, C₂-C₂₄ alkynyl, or C₁-C₂₄ acyl; and n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are different and are independently an optionallysubstituted C₄-C₂₀ alkyl, C₄-C₂₀ alkenyl, C₄-C₂₀ alkynyl, or C₄-C₂₀acyl.

In some embodiments, R⁴ is an optionally substituted C₁₂-C₂₄ alkyl,C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, and R⁵ is anoptionally substituted C₄-C₁₀ alkyl, C₄-C₁₀ alkenyl, C₄-C₁₀ alkynyl, orC₄-C₁₀ acyl. In certain instances, R⁴ is an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl, and R⁵ is an optionally substitutedC₄-C₈ or C₆ alkyl, C₄-C₈ or C₆ alkenyl, C₄-C₈ or C₆ alkynyl, or C₄-C₈ orC₆ acyl.

In other embodiments, R⁴ is an optionally substituted C₄-C₁₀ alkyl,C₄-C₁₀ alkenyl, C₄-C₁₀ alkynyl, or C₄-C₁₀ acyl, and R⁵ is an optionallysubstituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄acyl. In certain instances, R⁴ is an optionally substituted C₄-C₈ or C₆alkyl, C₄-C₈ or C₆ alkenyl, C₄-C₈ or C₆ alkynyl, or C₄-C₈ or C₆ acyl,and R⁵ is an optionally substituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ orC₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In particular embodiments, R⁴ is a linoleyl moiety, and R⁵ is a C₆ alkylmoiety, a C₆ alkenyl moiety, an octadecyl moiety, an oleyl moiety, alinolenyl moiety, a γ-linolenyl moiety, or a phytanyl moiety. In otherembodiments, one of R⁴ or R⁵ is a phytanyl moiety.

In some embodiments, the cationic lipid of Formula VII forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula VII is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of FormulaVII is an asymmetric lipid having a structure selected from the groupconsisting of:

In yet another aspect, cationic lipids of Formula VIII having thefollowing structure are useful in the present invention:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either the sameor different and are independently an optionally substituted C₁₂-C₂₄alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, wherein atleast one of R⁴ and R⁵ comprises at least four sites of unsaturation ora substituted C₁₂-C₂₄ alkyl; and n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In embodiments where at least one of R⁴ and R⁵ comprises a branchedalkyl group (e.g., a substituted C₁₂-C₂₄ alkyl group), the branchedalkyl group may comprise a C₁₂-C₂₄ alkyl having at least 1-6 (e.g., 1,2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. In particularembodiments, the branched alkyl group comprises a C₁₂-C₂₀ or C₁₄-C₂₂alkyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl,ethyl, propyl, or butyl) substituents. Preferably, the branched alkylgroup comprises a phytanyl (3,7,11,15-tetramethyl-hexadecanyl) moiety.

In alternative embodiments, at least one of R⁴ and R⁵ comprises abranched acyl group (e.g., a substituted C₁₂-C₂₄ acyl group). In certaininstances, the branched acyl group may comprise a C₁₂-C₂₄ acyl having atleast 1-6 (e.g., 1, 2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. Inparticular embodiments, the branched acyl group comprises a C₁₂-C₂₀ orC₁₄-C₂₂ acyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g.,methyl, ethyl, propyl, or butyl) substituents. Preferably, the branchedacyl group comprises a phytanoyl (3,7,11,15-tetramethyl-hexadecanoyl)moiety.

In embodiments where at least one of R⁴ and R⁵ comprises at least foursites of unsaturation, the double bonds present in one or both alkylchains may be in the cis and/or trans configuration. In a particularembodiment, R⁴ and R⁵ independently comprise four, five, or six sites ofunsaturation. In some instances, R⁴ comprises four, five, or six sitesof unsaturation and R⁵ comprises zero, one, two, three, four, five, orsix sites of unsaturation. In other instances, R⁴ comprises zero, one,two, three, four, five, or six sites of unsaturation and R⁵ comprisesfour, five, or six sites of unsaturation. In a preferred embodiment,both R⁴ and R⁵ comprise four, five, or six sites of unsaturation. Inparticular embodiments, R⁴ and R⁵ independently comprise a backbone offrom about 18 to about 24 carbon atoms, and one or both of R⁴ and R⁵independently comprise at least four, five, or six sites ofunsaturation.

In some embodiments, the cationic lipid of Formula VIII forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula VIII is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of FormulaVIII has a structure selected from the group consisting of:

In still yet another aspect, cationic lipids of Formula IX having thefollowing structure are useful in the present invention:

or salts thereof, wherein: R¹ is hydrogen (H) or —(CH₂)_(q)—NR⁶R⁷R⁸,wherein: R⁶ and R⁷ are either the same or different and areindependently an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, orC₂-C₆ alkynyl, or R⁶ and R⁷ may join to form an optionally substitutedheterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selectedfrom the group consisting of nitrogen (N), oxygen (O), and mixturesthereof; R⁸ is either absent or is hydrogen (H) or a C₁-C₆ alkyl toprovide a quaternary amine; and q is 0, 1, 2, 3, or 4; R² is anoptionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl; R³is either absent or is hydrogen (H) or a C₁-C₆ alkyl to provide aquaternary amine; R⁴ and R⁵ are either the same or different and areindependently an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl,C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and n is 0, 1, 2, 3, or 4.

In some embodiments, R² is an optionally substituted C₁-C₄ alkyl, C₂-C₄alkenyl, or C₂-C₄ alkynyl. In other embodiments, R³ is absent when thepH is above the pK_(a) of the cationic lipid and R³ is hydrogen when thepH is below the pK_(a) of the cationic lipid such that the amino headgroup is protonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In certainembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In further embodiments, R⁶ and R⁷ are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In otherembodiments, R⁸ is absent when the pH is above the pK_(a) of thecationic lipid and R⁸ is hydrogen when the pH is below the pK_(a) of thecationic lipid such that the amino head group is protonated. In analternative embodiment, R⁸ is an optionally substituted C₁-C₄ alkyl toprovide a quaternary amine.

In a preferred embodiment, R¹ is hydrogen and R² is an ethyl group. Inanother preferred embodiment, R⁶ and R⁷ are both methyl groups. Incertain instances, n is 1. In certain other instances, q is 1.

In certain embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, and abranched alkyl group as described above (e.g., a phytanyl moiety), aswell as acyl derivatives thereof (e.g., linoleoyl, linolenoyl,γ-linolenoyl, phytanoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In particularembodiments, R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties,γ-linolenyl moieties, or phytanyl moieties.

In some embodiments, the cationic lipid of Formula IX forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula IX is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula IXhas a structure selected from the group consisting of:

In another aspect, cationic lipids of Formula X having the followingstructure are useful in the present invention:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴, R⁵, and R⁶ are either thesame or different and are independently an optionally substitutedC₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; and nis 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, n is 1. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴, R⁵, and R⁶ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In certain embodiments, R⁴, R⁵, and R⁶ are independently selected fromthe group consisting of a dodecadienyl moiety, a tetradecadienyl moiety,a hexadecadienyl moiety, an octadecadienyl moiety, an icosadienylmoiety, a dodecatrienyl moiety, a tetradectrienyl moiety, ahexadecatrienyl moiety, an octadecatrienyl moiety, an icosatrienylmoiety, and a branched alkyl group as described above (e.g., a phytanylmoiety), as well as acyl derivatives thereof (e.g., linoleoyl,linolenoyl, γ-linolenoyl, phytanoyl, etc.). In some instances, theoctadecadienyl moiety is a linoleyl moiety. In other instances, theoctadecatrienyl moiety is a linolenyl moiety or a γ-linolenyl moiety. Inparticular embodiments, R⁴, R⁵, and R⁶ are all linoleyl moieties,linolenyl moieties, γ-linolenyl moieties, or phytanyl moieties.

In some embodiments, the cationic lipid of Formula X forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula X is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula Xhas a structure selected from the group consisting of:

In yet another aspect, cationic lipids of Formula XI having thefollowing structure are useful in the present invention:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either the sameor different and are independently an optionally substituted C₁₂-C₂₄alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; q is 0, 1, 2,3, or 4; and Y and Z are either the same or different and areindependently O, S, or NH, wherein if q is 1, R¹ and R² are both methylgroups, R⁴ and R⁵ are both linoleyl moieties, and Y and Z are both 0,then the alkylamino group is attached to one of the two carbons adjacentto Y or Z (i.e., at the ‘4’ or ‘6’ position of the 6-membered ring).

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, q is 2. In a particular embodiments, Y and Z are both oxygen(O). In other embodiments, R³ is absent when the pH is above the pK_(a)of the cationic lipid and R³ is hydrogen when the pH is below the pK_(a)of the cationic lipid such that the amino head group is protonated. Inan alternative embodiment, R³ is an optionally substituted C₁-C₄ alkylto provide a quaternary amine. In further embodiments, R⁴ and R⁵ areindependently an optionally substituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ orC₁₄-C₂₂ acyl.

In other embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, and abranched alkyl group as described above (e.g., a phytanyl moiety), aswell as acyl derivatives thereof (e.g., linoleoyl, linolenoyl,γ-linolenoyl, phytanoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In particularembodiments, R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties,γ-linolenyl moieties, or phytanyl moieties.

The alkylamino head group of Formula XI may be attached to the ‘4’ or‘5’ position of the 6-membered ring as shown below in an exemplaryembodiment wherein R¹ and R² are both methyl groups:

In further embodiments, the 6-membered ring of Formula XI may besubstituted with 1, 2, 3, 4, or 5 independently selected C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxyl, or hydroxyl substituents.In one particular embodiment, the 6-membered ring is substituted with 1,2, 3, 4, or 5 independently selected C₁-C₄ alkyl (e.g., methyl, ethyl,propyl, or butyl) substituents. An exemplary embodiment of a cationiclipid of Formula XI having a substituted 6-membered ring (methyl groupattached to the ‘4’ position) and wherein R¹ and R² are both methylgroups is shown below:

In particular embodiments, the cationic lipids of Formula XI may besynthesized using 2-hydroxymethyl-1,4-butanediol and 1,3,5-pentanetriol(or 3-methyl-1,3,5-pentanetriol) as starting materials.

In some embodiments, the cationic lipid of Formula XI forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula XI is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of Formula XIhas the structure:

In still yet another aspect, the present invention provides a cationiclipid of Formula XII having the following structure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either the sameor different and are independently an optionally substituted C₁₂-C₂₄alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl, wherein atleast one of R⁴ and R⁵ comprises at least one site of unsaturation inthe trans (K) configuration; m, n, and p are either the same ordifferent and are independently either 0, 1, or 2, with the proviso thatm, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Zare either the same or different and are independently O, S, or NH.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In another preferredembodiment, q is 2. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In certain embodiments, at least one of R⁴ and R⁵ further comprises one,two, three, four, five, six, or more sites of unsaturation in the cisand/or trans configuration. In some instances, R⁴ and R⁵ areindependently selected from any of the substituted or unsubstitutedalkyl or acyl groups described herein, wherein at least one or both ofR⁴ and R⁵ comprises at least one, two, three, four, five, or six sitesof unsaturation in the trans configuration. In one particularembodiment, R⁴ and R⁵ independently comprise a backbone of from about 12to about 22 carbon atoms (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,or 22 carbon atoms), and one or both of R⁴ and R⁵ independently compriseat least one, two, three, four, five, or six sites of unsaturation inthe trans configuration. In some preferred embodiments, at least one ofR⁴ and R⁵ comprises an (E)-heptadeceyl moiety. In other preferredembodiments, R⁴ and R⁵ are both (E)-8-heptadeceyl moieties.

In some embodiments, the cationic lipid of Formula XII forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula XII is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of FormulaXII has the structure:

In another aspect, the present invention provides a cationic lipid ofFormula XIII having the following structure:

or salts thereof, wherein: R¹ and R² are joined to form an optionallysubstituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2heteroatoms selected from the group consisting of nitrogen (N), oxygen(O), and mixtures thereof; R³ is either absent or is hydrogen (H) or aC₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either the sameor different and are independently an optionally substituted C₁₂-C₂₄alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; m, n, and pare either the same or different and are independently either 0, 1, or2, with the proviso that m, n, and p are not simultaneously 0; q is 0,1, 2, 3, or 4; and Y and Z are either the same or different and areindependently O, S, or NH.

In some embodiments, R¹ and R² are joined to form a heterocyclic ring of5 carbon atoms and 1 nitrogen atom. In certain instances, theheterocyclic ring is substituted with a substituent such as a hydroxylgroup at the ortho, meta, and/or para positions. In a preferredembodiment, q is 2. In other embodiments, R³ is absent when the pH isabove the pK_(a) of the cationic lipid and R³ is hydrogen when the pH isbelow the pK_(a) of the cationic lipid such that the amino head group isprotonated. In an alternative embodiment, R³ is an optionallysubstituted C₁-C₄ alkyl to provide a quaternary amine. In furtherembodiments, R⁴ and R⁵ are independently an optionally substitutedC₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl, C₁₂-C₂₀ or C₁₄-C₂₂alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl.

In certain embodiments, R⁴ and R⁵ are independently selected from thegroup consisting of a dodecadienyl moiety, a tetradecadienyl moiety, ahexadecadienyl moiety, an octadecadienyl moiety, an icosadienyl moiety,a dodecatrienyl moiety, a tetradectrienyl moiety, a hexadecatrienylmoiety, an octadecatrienyl moiety, an icosatrienyl moiety, and abranched alkyl group as described above (e.g., a phytanyl moiety), aswell as acyl derivatives thereof (e.g., linoleoyl, linolenoyl,γ-linolenoyl, phytanoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In other instances, the octadecatrienylmoiety is a linolenyl moiety or a γ-linolenyl moiety. In particularembodiments, R⁴ and R⁵ are both linoleyl moieties, linolenyl moieties,γ-linolenyl moieties, or phytanyl moieties.

In some embodiments, the cationic lipid of Formula XIII forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula XIII is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of FormulaXIII has the structure:

In yet another aspect, the present invention provides a cationic lipidof Formula XIV having the following structure:

or salts thereof, wherein:

-   -   R¹ and R² are either the same or different and are independently        an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆        alkynyl, or R¹ and R² may join to form an optionally substituted        heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms        selected from the group consisting of nitrogen (N), oxygen (O),        and mixtures thereof;    -   R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to        provide a quaternary amine;    -   R⁴ and R⁵ are either the same or different and are independently        a substituted C₁₂-C₂₄ alkyl; and    -   n is 0, 1, 2, 3, or 4.

In some embodiments, R¹ and R² are independently an optionallysubstituted C₁-C₄ alkyl, C₂-C₄ alkenyl, or C₂-C₄ alkynyl. In a preferredembodiment, R¹ and R² are both methyl groups. In one particularembodiment, n is 1. In another particular embodiment, n is 2. In otherembodiments, R³ is absent when the pH is above the pK_(a) of thecationic lipid and R³ is hydrogen when the pH is below the pK_(a) of thecationic lipid such that the amino head group is protonated. In analternative embodiment, R³ is an optionally substituted C₁-C₄ alkyl toprovide a quaternary amine.

In embodiments where at least one of R⁴ and R⁵ comprises a branchedalkyl group (e.g., a substituted C₁₂-C₂₄ alkyl group), the branchedalkyl group may comprise a C₁₂-C₂₄ alkyl having at least 1-6 (e.g., 1,2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. In particularembodiments, the branched alkyl group comprises a C₁₂-C₂₀ or C₁₄-C₂₂alkyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl,ethyl, propyl, or butyl) substituents. Preferably, the branched alkylgroup comprises a phytanyl (3,7,11,15-tetramethyl-hexadecanyl) moiety.In particular embodiments, R⁴ and R⁵ are both phytanyl moieties.

In alternative embodiments, at least one of R⁴ and R⁵ comprises abranched acyl group (e.g., a substituted C₁₂-C₂₄ acyl group). In certaininstances, the branched acyl group may comprise a C₁₂-C₂₄ acyl having atleast 1-6 (e.g., 1, 2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. Inparticular embodiments, the branched acyl group comprises a C₁₂-C₂₀ orC₁₄-C₂₂ acyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g.,methyl, ethyl, propyl, or butyl) substituents. Preferably, the branchedacyl group comprises a phytanoyl (3,7,11,15-tetramethyl-hexadecanoyl)moiety. In particular embodiments, R⁴ and R⁵ are both phytanoylmoieties.

In some embodiments, the cationic lipid of Formula XIV forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula XIV is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In a particularly preferred embodiment, the cationic lipid of FormulaXIV has a structure selected from the group consisting of:

The synthesis of cationic lipids of Formulas III-XIV is described hereinand in PCT Application No. PCT/CA2010/______, entitled “ImprovedCationic Lipids and Methods for the Delivery of Therapeutic Agents,”filed Jun. 30, 2010, bearing Attorney Docket No. 020801-009420PC, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

In some embodiments, a mixture of cationic lipids or salts thereof canbe included in the lipid particles of the present invention. In theseembodiments, the mixture of cationic lipids includes a cationic lipid ofFormulas I-XIV together with one or more additional cationic lipids.Other cationic lipids suitable for use in combination with the cationiclipids of Formulas I-XIV include cationic lipids of Formula XV havingthe following structure (or salts thereof):

wherein R¹ and R² are independently selected and are H or C₁-C₃ alkyls,R³ and R⁴ are independently selected and are alkyl groups having fromabout 10 to about 20 carbon atoms, and at least one of R³ and R⁴comprises at least two sites of unsaturation. In some instances, R¹ andR² are both methyl groups. In certain instances, R³ and R⁴ are both thesame, i.e., R³ and R⁴ are both linoleyl (C₁₈), etc. In other instances,R³ and R⁴ are different, i.e., R³ is tetradectrienyl (C₁₄) and R⁴ islinoleyl (C₁₈). In a preferred embodiment, the cationic lipid of FormulaXV is symmetrical, i.e., R³ and R⁴ are both the same. In anotherpreferred embodiment, both R³ and R⁴ comprise at least two sites ofunsaturation. In some embodiments, R³ and R⁴ are independently selectedfrom the group consisting of dodecadienyl, tetradecadienyl,hexadecadienyl, linoleyl, and icosadienyl. In a preferred embodiment, R³and R⁴ are both linoleyl. In some embodiments, R³ and R⁴ comprise atleast three sites of unsaturation and are independently selected from,e.g., dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, andicosatrienyl. In particular embodiments, the cationic lipid of FormulaXV comprises 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), or mixturesthereof.

In some embodiments, the cationic lipid of Formula XV forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula XV is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

Other cationic lipids suitable for use in combination with the cationiclipids of Formulas I-XIV include cationic lipids of Formula XVI havingthe following structure (or salts thereof):

wherein R¹ and R² are independently selected and are H or C₁-C₃ alkyls,R³ and R⁴ are independently selected and are alkyl groups having fromabout 10 to about 20 carbon atoms, and at least one of R³ and R⁴comprises at least two sites of unsaturation. In certain instances, R³and R⁴ are both the same, i.e., R³ and R⁴ are both linoleyl (C₁₈), etc.In certain other instances, R³ and R⁴ are different, i.e., R³ istetradectrienyl (C₁₄) and R⁴ is linoleyl (C₁₈). In a preferredembodiment, the cationic lipid of Formula XVI is symmetrical, i.e., R³and R⁴ are both the same. In another preferred embodiment, both R³ andR⁴ comprise at least two sites of unsaturation. In some embodiments, R³and R⁴ are independently selected from the group consisting ofdodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, andicosadienyl. In a preferred embodiment, R³ and R⁴ are both linoleyl. Insome embodiments, R³ and R⁴ comprise at least three sites ofunsaturation and are independently selected from, e.g., dodecatrienyl,tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

In some embodiments, the cationic lipid of Formula XVI forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula XVI is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of cationic lipids such as DLinDMA and DLenDMA, as well asadditional cationic lipids falling within the scope of Formulas XV andXVI, is described in U.S. Patent Publication No. 20060083780, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

In addition to the cationic lipids of Formulas XV-XVI, other cationiclipids suitable for use in combination with one or more cationic lipidsof Formulas I-XIV include, but are not limited to,1,2-dioeylcarbamoyloxy-3-dimethylaminopropane (DO-C-DAP),1,2-dimyristoleoyl-3-dimethylaminopropane (DMDAP),1,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.Cl),dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-K-DMA; also known asDLin-M-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),and mixtures thereof.

Additional cationic lipids suitable for use in combination with one ormore cationic lipids of Formulas I-XIV include, without limitation,cationic lipids such as(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA or “MC3”) and certain analogs thereof asdescribed in U.S. Provisional Patent Application No. 61/334,104,entitled “Novel Cationic Lipids and Methods of Use Thereof,” filed May12, 2010, and PCT Publication Nos. WO 2010/054401, WO 2010/054405, WO2010/054406, and WO 2010/054384, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

The synthesis of cationic lipids such as DO-C-DAP, DMDAP, DOTAP.Cl,DLin-M-K-DMA, as well as additional cationic lipids, is described in PCTPublication No. WO 2010/042877, the disclosure of which is incorporatedherein by reference in its entirety for all purposes.

The synthesis of cationic lipids such as DLin-C-DAP, DLinDAC, DLinMA,DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLinTMA.Cl, DLinTAP.Cl, DLinMPZ,DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, isdescribed in PCT Publication No. WO 09/086,558, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

The synthesis of cationic lipids such as CLinDMA, as well as additionalcationic lipids, is described in U.S. Patent Publication No.20060240554, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The synthesis of a number of other cationic lipids and related analogshas been described in U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833;5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO96/10390, the disclosures of which are each herein incorporated byreference in their entirety for all purposes. Additionally, a number ofcommercial preparations of cationic lipids can be used, such as, e.g.,LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO/BRL);LIPOFECTAMINE® (including DOSPA and DOPE, available from GIBCO/BRL); andTRANSFECTAM® (including DOGS, available from Promega Corp.).

In some embodiments, the cationic lipid comprises from about 45 mol % toabout 90 mol %, from about 45 mol % to about 85 mol %, from about 45 mol% to about 80 mol %, from about 45 mol % to about 75 mol %, from about45 mol % to about 70 mol %, from about 45 mol % to about 65 mol %, fromabout 45 mol % to about 60 mol %, from about 45 mol % to about 55 mol %,from about 50 mol % to about 90 mol %, from about 50 mol % to about 85mol %, from about 50 mol % to about 80 mol %, from about 50 mol % toabout 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol% to about 65 mol %, from about 50 mol % to about 60 mol %, from about55 mol % to about 65 mol % or from about 55 mol % to about 70 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle.

In certain preferred embodiments, the cationic lipid comprises fromabout 50 mol % to about 58 mol %, from about 51 mol % to about 59 mol %,from about 51 mol % to about 58 mol %, from about 51 mol % to about 57mol %, froth about 52 mol % to about 58 mol %, from about 52 mol % toabout 57 mol %, from about 52 mol % to about 56 mol %, or from about 53mol % to about 55 mol % (or any fraction thereof or range therein) ofthe total lipid present in the particle. In particular embodiments, thecationic lipid comprises about 50 mol %, 51 mol %, 52 mol %, 53 mol %,54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60 mol %, 61mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (or any fractionthereof or range therein) of the total lipid present in the particle. Incertain other embodiments, the cationic lipid comprises (at least) about66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, or 90 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In additional embodiments, the cationic lipid comprises from about 2 mol% to about 60 mol %, from about 5 mol % to about 50 mol %, from about 10mol % to about 50 mol %, from about 20 mol % to about 50 mol %, fromabout 20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %,or about 40 mol % (or any fraction thereof or range therein) of thetotal lipid present in the particle.

Additional percentages and ranges of cationic lipids suitable for use inthe lipid particles of the present invention are described in PCTPublication No. WO 09/127,060, U.S. application Ser. No. 12/794,701,filed Jun. 4, 2010, PCT Application No. PCT/CA2010/______, entitled“Improved Cationic Lipids and Methods for the Delivery of TherapeuticAgents,” filed Jun. 30, 2010, bearing Attorney Docket No.020801-009420PC, and U.S. application Ser. No. ______, entitled “NovelLipid Formulations for Delivery of Therapeutic Agents to Solid Tumors,”filed Jun. 30, 2010, bearing Attorney Docket No. 020801-009610US, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

It should be understood that the percentage of cationic lipid present inthe lipid particles of the invention is a target amount, and that theactual amount of cationic lipid present in the formulation may vary, forexample, by ±5 mol %. For example, in the 1:57 lipid particle (e.g.,SNALP) formulation, the target amount of cationic lipid is 57.1 mol %,but the actual amount of cationic lipid may be ±5 mol %, ±4 mol %, ±3mol %, ±2 mol %, ±1 mol %, t 0.75 mol %, ±0.5 mol %, t 0.25 mol %, or±0.1 mol % of that target amount, with the balance of the formulationbeing made up of other lipid components (adding up to 100 mol % of totallipids present in the particle). Similarly, in the 7:54 lipid particle(e.g., SNALP) formulation, the target amount of cationic lipid is 54.06mol %, but the actual amount of cationic lipid may be ±5 mol %, ±4 mol%, ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %,or ±0.1 mol % of that target amount, with the balance of the formulationbeing made up of other lipid components (adding up to 100 mol % of totallipids present in the particle).

C. Non-Cationic Lipids

The non-cationic lipids used in the lipid particles of the invention(e.g., SNALP) can be any of a variety of neutral uncharged,zwitterionic, or anionic lipids capable of producing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof. Non-limiting examples ofcholesterol derivatives include polar analogues such as 5α-cholestanol,5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether,cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polaranalogues such as 5α-cholestane, cholestenone, 5α-cholestanone,5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. Inpreferred embodiments, the cholesterol derivative is a polar analoguesuch as cholesteryl-(4′-hydroxy)-butyl ether. The synthesis ofcholesteryl-(2′-hydroxy)-ethyl ether is described in PCT Publication No.WO 09/127,060, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

In some embodiments, the non-cationic lipid present in the lipidparticles (e.g., SNALP) comprises or consists of a mixture of one ormore phospholipids and cholesterol or a derivative thereof. In otherembodiments, the non-cationic lipid present in the lipid particles(e.g., SNALP) comprises or consists of one or more phospholipids, e.g.,a cholesterol-free lipid particle formulation. In yet other embodiments,the non-cationic lipid present in the lipid particles (e.g., SNALP)comprises or consists of cholesterol or a derivative thereof, e.g., aphospholipid-free lipid particle formulation.

Other examples of non-cationic lipids suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

In some embodiments, the non-cationic lipid comprises from about 10 mol% to about 60 mol %, from about 20 mol % to about 55 mol %, from about20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, fromabout 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %,from about 30 mol % to about 50 mol %, from about 30 mol % to about 45mol %, from about 30 mol % to about 40 mol %, from about 35 mol % toabout 45 mol %, from about 37 mol % to about 42 mol %, or about 35 mol%, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %,43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In embodiments where the lipid particles contain a mixture ofphospholipid and cholesterol or a cholesterol derivative, the mixturemay comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60mol % of the total lipid present in the particle.

In some embodiments, the phospholipid component in the mixture maycomprise from about 2 mol % to about 20 mol %, from about 2 mol % toabout 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol %to about 15 mol %, or from about 4 mol % to about 10 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the phospholipid componentin the mixture comprises from about 5 mol % to about 10 mol %, fromabout 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %,from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol%, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. As a non-limiting example, a 1:57 lipid particle formulationcomprising a mixture of phospholipid and cholesterol may comprise aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof), e.g., in a mixture with cholesterol or a cholesterolderivative at about 34 mol % (or any fraction thereof) of the totallipid present in the particle. As another non-limiting example, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise a phospholipid such as DPPC or DSPC at about 7mol % (or any fraction thereof), e.g., in a mixture with cholesterol ora cholesterol derivative at about 32 mol % (or any fraction thereof) ofthe total lipid present in the particle.

In other embodiments, the cholesterol component in the mixture maycomprise from about 25 mol % to about 45 mol %, from about 25 mol % toabout 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol% to about 40 mol %, from about 27 mol % to about 37 mol %, from about25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the cholesterol component inthe mixture comprises from about 25 mol % to about 35 mol %, from about27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, fromabout 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %,from about 31 mol % to about 33 mol %, or about 30 mol %, 31 mol %, 32mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle. In otherembodiments, the cholesterol component in the mixture comprises about36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mol % (or any fraction thereofor range therein) of the total lipid present in the particle. Typically,a 1:57 lipid particle formulation comprising a mixture of phospholipidand cholesterol may comprise cholesterol or a cholesterol derivative atabout 34 mol % (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle. Typically, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise cholesterol or a cholesterol derivative atabout 32 mol % (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle.

In embodiments where the lipid particles are phospholipid-free, thecholesterol or derivative thereof may comprise up to about 25 mol %, 30mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % ofthe total lipid present in the particle.

In some embodiments, the cholesterol or derivative thereof in thephospholipid-free lipid particle formulation may comprise from about 25mol % to about 45 mol %, from about 25 mol % to about 40 mol %, fromabout 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %,from about 31 mol % to about 39 mol %, from about 32 mol % to about 38mol %, from about 33 mol % to about 37 mol %, from about 35 mol % toabout 45 mol %, from about 30 mol % to about 35 mol %, from about 35 mol% to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41mol %, 42 mol %, 43 mol %, 44 mol %, or 45 mol % (or any fractionthereof or range therein) of the total lipid present in the particle. Asa non-limiting example, a 1:62 lipid particle formulation may comprisecholesterol at about 37 mol % (or any fraction thereof) of the totallipid present in the particle. As another non-limiting example, a 7:58lipid particle formulation may comprise cholesterol at about 35 mol %(or any fraction thereof) of the total lipid present in the particle.

In other embodiments, the non-cationic lipid comprises from about 5 mol% to about 90 mol %, from about 10 mol % to about 85 mol %, from about20 mol % to about 80 mol %, about 10 mol % (e.g., phospholipid only), orabout 60 mol % (e.g., phospholipid and cholesterol or derivativethereof) (or any fraction thereof or range therein) of the total lipidpresent in the particle.

Additional percentages and ranges of non-cationic lipids suitable foruse in the lipid particles of the present invention are described in PCTPublication No. WO 09/127,060, U.S. application Ser. No. 12/794,701,filed Jun. 4, 2010, PCT Application No. PCT/CA2010/______, entitled“Improved Cationic Lipids and Methods for the Delivery of TherapeuticAgents,” filed Jun. 30, 2010, bearing Attorney Docket No.020801-009420PC, and U.S. application Ser. No. ______, entitled “NovelLipid Formulations for Delivery of Therapeutic Agents to Solid Tumors,”filed Jun. 30, 2010, bearing Attorney Docket No. 020801-009610US, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

It should be understood that the percentage of non-cationic lipidpresent in the lipid particles of the invention is a target amount, andthat the actual amount of non-cationic lipid present in the formulationmay vary, for example, by ±5 mol %. For example, in the 1:57 lipidparticle (e.g., SNALP) formulation, the target amount of phospholipid is7.1 mol % and the target amount of cholesterol is 34.3 mol %, but theactual amount of phospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that targetamount, and the actual amount of cholesterol may be ±3 mol %, ±2 mol %,±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle). Similarly, in the 7:54 lipid particle (e.g., SNALP)formulation, the target amount of phospholipid is 6.75 mol % and thetarget amount of cholesterol is 32.43 mol %, but the actual amount ofphospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %, ±0.75 mol %, ±0.5mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, and the actualamount of cholesterol may be ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, with thebalance of the formulation being made up of other lipid components(adding up to 100 mol % of total lipids present in the particle).

D. Lipid Conjugates

In addition to cationic and non-cationic lipids, the lipid particles ofthe invention (e.g., SNALP) may further comprise a lipid conjugate. Theconjugated lipid is useful in that it prevents the aggregation ofparticles. Suitable conjugated lipids include, but are not limited to,PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates,cationic-polymer-lipid conjugates (CPLs), and mixtures thereof. Incertain embodiments, the particles comprise either a PEG-lipid conjugateor an ATTA-lipid conjugate together with a CPL.

In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examplesof PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEGcoupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613,PEG conjugated to cholesterol or a derivative thereof, and mixturesthereof. The disclosures of these patent documents are hereinincorporated by reference in their entirety for all purposes.

Additional PEG-lipids suitable for use in the invention include, withoutlimitation, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG).The synthesis of PEG-C-DOMG is described in PCT Publication No. WO09/086,558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. Yet additional suitable PEG-lipidconjugates include, without limitation,1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-ω-methyl-poly(ethyleneglycol) (2 KPEG-DMG). The synthesis of 2 KPEG-DMG is described in U.S.Pat. No. 7,404,969, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, but are not limited to, thefollowing: monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S-NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES),monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as wellas such compounds containing a terminal hydroxyl group instead of aterminal methoxy group (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH₂, etc.).Other PEGs such as those described in U.S. Pat. Nos. 6,774,180 and7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing thePEG-lipid conjugates of the present invention. The disclosures of thesepatents are herein incorporated by reference in their entirety for allpurposes. In addition, monomethoxypolyethyleneglycol-acetic acid(MePEG-CH₂COOH) is particularly useful for preparing PEG-lipidconjugates including, e.g., PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). In otherinstances, the PEG moiety has an average molecular weight of from about550 daltons to about 1000 daltons, from about 250 daltons to about 1000daltons, from about 400 daltons to about 1000 daltons, from about 600daltons to about 900 daltons, from about 700 daltons to about 800daltons, or about 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, or 1000 daltons. In preferred embodiments, thePEG moiety has an average molecular weight of about 2,000 daltons orabout 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skilled in the art.Phosphatidyl-ethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

The term “ATTA” or “polyamide” includes, without limitation, compoundsdescribed in U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. These compounds include a compound having the formula:

wherein R is a member selected from the group consisting of hydrogen,alkyl and acyl; R¹ is a member selected from the group consisting ofhydrogen and alkyl; or optionally, R and R¹ and the nitrogen to whichthey are bound form an azido moiety; R² is a member of the groupselected from hydrogen, optionally substituted alkyl, optionallysubstituted aryl and a side chain of an amino acid; R³ is a memberselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, mercapto, hydrazino, amino and NR⁴R⁵, wherein R⁴ and R⁵ areindependently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4;and q is 0 or 1. It will be apparent to those of skill in the art thatother polyamides can be used in the compounds of the present invention.

The term “diacylglycerol” or “DAG” includes a compound having 2 fattyacyl chains, R¹ and R², both of which have independently between 2 and30 carbons bonded to the 1- and 2-position of glycerol by esterlinkages. The acyl groups can be saturated or have varying degrees ofunsaturation. Suitable acyl groups include, but are not limited to,lauroyl (C12), myristoyl (C₁₄), palmitoyl (C₁₆), stearoyl (C₁₈), andicosoyl (C₂₀). In preferred embodiments, R¹ and R² are the same, i.e.,R¹ and R² are both myristoyl (i.e., dimyristoyl), R¹ and R² are bothstearoyl (i.e., distearoyl), etc. Diacylglycerols have the followinggeneral formula:

The term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkylchains, R¹ and R², both of which have independently between 2 and 30carbons. The alkyl groups can be saturated or have varying degrees ofunsaturation. Dialkyloxypropyls have the following general formula:

In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate havingthe following formula:

wherein R¹ and R² are independently selected and are long-chain alkylgroups having from about 10 to about 22 carbon atoms; PEG is apolyethyleneglycol; and L is a non-ester containing linker moiety or anester containing linker moiety as described above. The long-chain alkylgroups can be saturated or unsaturated. Suitable alkyl groups include,but are not limited to, decyl (C₁₀), lauryl (C₁₂), myristyl (C₁₄),palmityl (C₁₆), stearyl (C₁₈), and icosyl (C20). In preferredembodiments, R¹ and R² are the same, i.e., R¹ and R² are both myristyl(i.e., dimyristyl), R¹ and R² are both stearyl (i.e., distearyl), etc.

In Formula XX above, the PEG has an average molecular weight rangingfrom about 550 daltons to about 10,000 daltons. In certain instances,the PEG has an average molecular weight of from about 750 daltons toabout 5,000 daltons (e.g., from about 1,000 daltons to about 5,000daltons, from about 1,500 daltons to about 3,000 daltons, from about 750daltons to about 3,000 daltons, from about 750 daltons to about 2,000daltons, etc.). In other instances, the PEG moiety has an averagemolecular weight of from about 550 daltons to about 1000 daltons, fromabout 250 daltons to about 1000 daltons, from about 400 daltons to about1000 daltons, from about 600 daltons to about 900 daltons, from about700 daltons to about 800 daltons, or about 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 daltons. Inpreferred embodiments, the PEG has an average molecular weight of about2,000 daltons or about 750 daltons. The PEG can be optionallysubstituted with alkyl, alkoxy, acyl, or aryl groups. In certainembodiments, the terminal hydroxyl group is substituted with a methoxyor methyl group.

In a preferred embodiment, “L” is a non-ester containing linker moiety.Suitable non-ester containing linkers include, but are not limited to,an amido linker moiety, an amino linker moiety, a carbonyl linkermoiety, a carbamate linker moiety, a urea linker moiety, an ether linkermoiety, a disulphide linker moiety, a succinamidyl linker moiety, andcombinations thereof. In a preferred embodiment, the non-estercontaining linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAAconjugate). In another preferred embodiment, the non-ester containinglinker moiety is an amido linker moiety (i.e., a PEG-A-DAA conjugate).In yet another preferred embodiment, the non-ester containing linkermoiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).

In particular embodiments, the PEG-lipid conjugate is selected from:

The PEG-DAA conjugates are synthesized using standard techniques andreagents known to those of skill in the art. It will be recognized thatthe PEG-DAA conjugates will contain various amide, amine, ether, thio,carbamate, and urea linkages. Those of skill in the art will recognizethat methods and reagents for forming these bonds are well known andreadily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); andFurniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed.(Longman 1989). It will also be appreciated that any functional groupspresent may require protection and deprotection at different points inthe synthesis of the PEG-DAA conjugates. Those of skill in the art willrecognize that such techniques are well known. See, e.g., Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C₁₀)conjugate, a PEG-dilauryloxypropyl (C₁₂) conjugate, aPEG-dimyristyloxypropyl (C₁₄) conjugate, a PEG-dipalmityloxypropyl (C₁₆)conjugate, or a PEG-distearyloxypropyl (C₁₈) conjugate. In theseembodiments, the PEG preferably has an average molecular weight of about750 or about 2,000 daltons. In one particularly preferred embodiment,the PEG-lipid conjugate comprises PEG2000-C-DMA, wherein the “2000”denotes the average molecular weight of the PEG, the “C” denotes acarbamate linker moiety, and the “DMA” denotes dimyristyloxypropyl. Inanother particularly preferred embodiment, the PEG-lipid conjugatecomprises PEG750-C-DMA, wherein the “750” denotes the average molecularweight of the PEG, the “C” denotes a carbamate linker moiety, and the“DMA” denotes dimyristyloxypropyl. In particular embodiments, theterminal hydroxyl group of the PEG is substituted with a methyl group.Those of skill in the art will readily appreciate that otherdialkyloxypropyls can be used in the PEG-DAA conjugates of the presentinvention.

In addition to the foregoing, it will be readily apparent to those ofskill in the art that other hydrophilic polymers can be used in place ofPEG. Examples of suitable polymers that can be used in place of PEGinclude, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

In addition to the foregoing components, the lipid particles (e.g.,SNALP) of the present invention can further comprise cationicpoly(ethylene glycol) (PEG) lipids or CPLs (see, e.g., Chen et al.,Bioconj. Chem., 11:433-437 (2000); U.S. Pat. No. 6,852,334; PCTPublication No. WO 00/62813, the disclosures of which are hereinincorporated by reference in their entirety for all purposes).

Suitable CPLs include compounds of Formula XXI:

A-W-Y  (XXI),

wherein A, W, and Y are as described below.

With reference to Formula XXI, “A” is a lipid moiety such as anamphipathic lipid, a neutral lipid, or a hydrophobic lipid that acts asa lipid anchor. Suitable lipid examples include, but are not limited to,diacylglycerolyls, dialkylglycerolyls, N—N-dialkylaminos,1,2-diacyloxy-3-aminopropanes, and 1,2-dialkyl-3-aminopropanes.

“W” is a polymer or an oligomer such as a hydrophilic polymer oroligomer. Preferably, the hydrophilic polymer is a biocompatable polymerthat is nonimmunogenic or possesses low inherent immunogenicity.Alternatively, the hydrophilic polymer can be weakly antigenic if usedwith appropriate adjuvants. Suitable nonimmunogenic polymers include,but are not limited to, PEG, polyamides, polylactic acid, polyglycolicacid, polylactic acid/polyglycolic acid copolymers, and combinationsthereof. In a preferred embodiment, the polymer has a molecular weightof from about 250 to about 7,000 daltons.

“Y” is a polycationic moiety. The term polycationic moiety refers to acompound, derivative, or functional group having a positive charge,preferably at least 2 positive charges at a selected pH, preferablyphysiological pH. Suitable polycationic moieties include basic aminoacids and their derivatives such as arginine, asparagine, glutamine,lysine, and histidine; spermine; spermidine; cationic dendrimers;polyamines; polyamine sugars; and amino polysaccharides. Thepolycationic moieties can be linear, such as linear tetralysine,branched or dendrimeric in structure. Polycationic moieties have betweenabout 2 to about 15 positive charges, preferably between about 2 toabout 12 positive charges, and more preferably between about 2 to about8 positive charges at selected pH values. The selection of whichpolycationic moiety to employ may be determined by the type of particleapplication which is desired.

The charges on the polycationic moieties can be either distributedaround the entire particle moiety, or alternatively, they can be adiscrete concentration of charge density in one particular area of theparticle moiety e.g., a charge spike. If the charge density isdistributed on the particle, the charge density can be equallydistributed or unequally distributed. All variations of chargedistribution of the polycationic moiety are encompassed by the presentinvention.

The lipid “A” and the nonimmunogenic polymer “W” can be attached byvarious methods and preferably by covalent attachment. Methods known tothose of skill in the art can be used for the covalent attachment of “A”and “W.” Suitable linkages include, but are not limited to, amide,amine, carboxyl, carbonate, carbamate, ester, and hydrazone linkages. Itwill be apparent to those skilled in the art that “A” and “W” must havecomplementary functional groups to effectuate the linkage. The reactionof these two groups, one on the lipid and the other on the polymer, willprovide the desired linkage. For example, when the lipid is adiacylglycerol and the terminal hydroxyl is activated, for instance withNHS and DCC, to form an active ester, and is then reacted with a polymerwhich contains an amino group, such as with a polyamide (see, e.g., U.S.Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which are hereinincorporated by reference in their entirety for all purposes), an amidebond will form between the two groups.

In certain instances, the polycationic moiety can have a ligandattached, such as a targeting ligand or a chelating moiety forcomplexing calcium. Preferably, after the ligand is attached, thecationic moiety maintains a positive charge. In certain instances, theligand that is attached has a positive charge. Suitable ligands include,but are not limited to, a compound or device with a reactive functionalgroup and include lipids, amphipathic lipids, carrier compounds,bioaffinity compounds, biomaterials, biopolymers, biomedical devices,analytically detectable compounds, therapeutically active compounds,enzymes, peptides, proteins, antibodies, immune stimulators,radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA,polysaccharides, liposomes, virosomes, micelles, immunoglobulins,functional groups, other targeting moieties, or toxins.

In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % toabout 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8mol % to about 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, fromabout 0.9 mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol%, from about 1 mol % to about 1.7 mol %, from about 1.2 mol % to about1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol %to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, or about 1,1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or any fractionthereof or range therein) of the total lipid present in the particle.

In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % toabout 18 mol %, from about 2 mol % to about 15 mol %, from about 4 mol %to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5mol % to about 12 mol %, or about 2 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle.

In further embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol%, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol% (or any fraction thereof or range therein) of the total lipid presentin the particle.

Additional examples, percentages, and/or ranges of lipid conjugatessuitable for use in the lipid particles of the invention are describedin PCT Publication No. WO 09/127,060, U.S. application Ser. No.12/794,701, filed Jun. 4, 2010, PCT Application No. PCT/CA2010/______,entitled “Improved Cationic Lipids and Methods for the Delivery ofTherapeutic Agents,” filed Jun. 30, 2010, bearing Attorney Docket No.020801-009420PC, U.S. application Ser. No. ______, entitled “Novel LipidFormulations for Delivery of Therapeutic Agents to Solid Tumors,” filedJun. 30, 2010, bearing Attorney Docket No. 020801-009610US, U.S.Provisional Application No. 61/294,828, filed Jan. 13, 2010, U.S.Provisional Application No. 61/295, 140, filed Jan. 14, 2010, and PCTPublication No. WO 2010/006282, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of lipid conjugate (e.g.,PEG-lipid) present in the lipid particles of the invention is a targetamount, and that the actual amount of lipid conjugate present in theformulation may vary, for example, by ±2 mol %. For example, in the 1:57lipid particle (e.g., SNALP) formulation, the target amount of lipidconjugate is 1.4 mol %, but the actual amount of lipid conjugate may be±0.5 mol %, ±0.4 mol %, ±0.3 mol %, ±0.2 mol %, ±0.1 mol %, or ±0.05 mol% of that target amount, with the balance of the formulation being madeup of other lipid components (adding up to 100 mol % of total lipidspresent in the particle). Similarly, in the 7:54 lipid particle (e.g.,SNALP) formulation, the target amount of lipid conjugate is 6.76 mol %,but the actual amount of lipid conjugate may be ±2 mol %, ±1.5 mol %, ±1mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle).

One of ordinary skill in the art will appreciate that the concentrationof the lipid conjugate can be varied depending on the lipid conjugateemployed and the rate at which the lipid particle is to becomefusogenic.

By controlling the composition and concentration of the lipid conjugate,one can control the rate at which the lipid conjugate exchanges out ofthe lipid particle and, in turn, the rate at which the lipid particlebecomes fusogenic. For instance, when a PEG-DAA conjugate is used as thelipid conjugate, the rate at which the lipid particle becomes fusogeniccan be varied, for example, by varying the concentration of the lipidconjugate, by varying the molecular weight of the PEG, or by varying thechain length and degree of saturation of the alkyl groups on the PEG-DAAconjugate. In addition, other variables including, for example, pH,temperature, ionic strength, etc. can be used to vary and/or control therate at which the lipid particle becomes fusogenic. Other methods whichcan be used to control the rate at which the lipid particle becomesfusogenic will become apparent to those of skill in the art upon readingthis disclosure. Also, by controlling the composition and concentrationof the lipid conjugate, one can control the lipid particle (e.g., SNALP)size.

V. Preparation of Lipid Particles

The lipid particles of the present invention, e.g., SNALP, in which anactive agent or therapeutic agent such as an interfering RNA (e.g.,siRNA) is entrapped within the lipid portion of the particle and isprotected from degradation, can be formed by any method known in the artincluding, but not limited to, a continuous mixing method, a directdilution process, and an in-line dilution process.

In particular embodiments, the cationic lipids may comprise lipids ofFormulas I-XIV or salts thereof, alone or in combination with othercationic lipids. In other embodiments, the non-cationic lipids are eggsphingomyelin (ESM), distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC),1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC),dipalmitoyl-phosphatidylcholine (DPPC),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,14:0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE(1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE(1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE(1,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1 trans PE(1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE(1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethyleneglycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modifieddiacylglycerols, or PEG-modified dialkyloxypropyls), cholesterol,derivatives thereof, or combinations thereof.

In certain embodiments, the present invention provides nucleicacid-lipid particles (e.g., SNALP) produced via a continuous mixingmethod, e.g., a process that includes providing an aqueous solutioncomprising a nucleic acid (e.g., interfering RNA) in a first reservoir,providing an organic lipid solution in a second reservoir (wherein thelipids present in the organic lipid solution are solubilized in anorganic solvent, e.g., a lower alkanol such as ethanol), and mixing theaqueous solution with the organic lipid solution such that the organiclipid solution mixes with the aqueous solution so as to substantiallyinstantaneously produce a lipid vesicle (e.g., liposome) encapsulatingthe nucleic acid within the lipid vesicle. This process and theapparatus for carrying out this process are described in detail in U.S.Patent Publication No. 20040142025, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The action of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a lipid vesicle substantially instantaneously upon mixing. Asused herein, the phrase “continuously diluting a lipid solution with abuffer solution” (and variations) generally means that the lipidsolution is diluted sufficiently rapidly in a hydration process withsufficient force to effectuate vesicle generation. By mixing the aqueoussolution comprising a nucleic acid with the organic lipid solution, theorganic lipid solution undergoes a continuous stepwise dilution in thepresence of the buffer solution (i.e., aqueous solution) to produce anucleic acid-lipid particle.

The nucleic acid-lipid particles formed using the continuous mixingmethod typically have a size of from about 30 nm to about 150 nm, fromabout 40 nm to about 150 nm, from about 50 nm to about 150 nm, fromabout 60 nm to about 130 nm, from about 70 nm to about 110 nm, fromabout 70 nm to about 100 nm, from about 80 nm to about 100 nm, fromabout 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, or 150 nm (or any fraction thereof or range therein). Theparticles thus formed do not aggregate and are optionally sized toachieve a uniform particle size.

In another embodiment, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) produced via a direct dilution process thatincludes forming a lipid vesicle (e.g., liposome) solution andimmediately and directly introducing the lipid vesicle solution into acollection vessel containing a controlled amount of dilution buffer. Inpreferred aspects, the collection vessel includes one or more elementsconfigured to stir the contents of the collection vessel to facilitatedilution. In one aspect, the amount of dilution buffer present in thecollection vessel is substantially equal to the volume of lipid vesiclesolution introduced thereto. As a non-limiting example, a lipid vesiclesolution in 45% ethanol when introduced into the collection vesselcontaining an equal volume of dilution buffer will advantageously yieldsmaller particles.

In yet another embodiment, the present invention provides nucleicacid-lipid particles (e.g., SNALP) produced via an in-line dilutionprocess in which a third reservoir containing dilution buffer is fluidlycoupled to a second mixing region. In this embodiment, the lipid vesicle(e.g., liposome) solution formed in a first mixing region is immediatelyand directly mixed with dilution buffer in the second mixing region. Inpreferred aspects, the second mixing region includes a T-connectorarranged so that the lipid vesicle solution and the dilution bufferflows meet as opposing 180° flows; however, connectors providingshallower angles can be used, e.g., from about 27° to about 180° (e.g.,about 90°). A pump mechanism delivers a controllable flow of buffer tothe second mixing region. In one aspect, the flow rate of dilutionbuffer provided to the second mixing region is controlled to besubstantially equal to the flow rate of lipid vesicle solutionintroduced thereto from the first mixing region. This embodimentadvantageously allows for more control of the flow of dilution buffermixing with the lipid vesicle solution in the second mixing region, andtherefore also the concentration of lipid vesicle solution in bufferthroughout the second mixing process. Such control of the dilutionbuffer flow rate advantageously allows for small particle size formationat reduced concentrations.

These processes and the apparatuses for carrying out these directdilution and in-line dilution processes are described in detail in U.S.Patent Publication No. 20070042031, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The nucleic acid-lipid particles formed using the direct dilution andin-line dilution processes typically have a size of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, from about 70 nm to about 100 nm, from about 80 nm toabout 100 nm, from about 90 nm to about 100 nm, from about 70 to about90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm,less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm,35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130nm, 135 nm, 140 nm, 145 nm, or 150 nm (or any fraction thereof or rangetherein). The particles thus formed do not aggregate and are optionallysized to achieve a uniform particle size.

If needed, the lipid particles of the invention (e.g., SNALP) can besized by any of the methods available for sizing liposomes. The sizingmay be conducted in order to achieve a desired size range and relativelynarrow distribution of particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles, is described in U.S. Pat. No. 4,737,323, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. Sonicating a particle suspension either by bath orprobe sonication produces a progressive size reduction down to particlesof less than about 50 nm in size. Homogenization is another method whichrelies on shearing energy to fragment larger particles into smallerones. In a typical homogenization procedure, particles are recirculatedthrough a standard emulsion homogenizer until selected particle sizes,typically between about 60 and about 80 nm, are observed. In bothmethods, the particle size distribution can be monitored by conventionallaser-beam particle size discrimination, or QELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In some embodiments, the nucleic acids present in the particles areprecondensed as described in, e.g., U.S. patent application Ser. No.09/744,103, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

In other embodiments, the methods may further comprise adding non-lipidpolycations which are useful to effect the lipofection of cells usingthe present compositions. Examples of suitable non-lipid polycationsinclude, hexadimethrine bromide (sold under the brand name POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine, and polyethyleneimine. Addition of these salts ispreferably after the particles have been formed.

In some embodiments, the nucleic acid to lipid ratios (mass/mass ratios)in a formed nucleic acid-lipid particle (e.g., SNALP) will range fromabout 0.01 to about 0.2, from about 0.05 to about 0.2, from about 0.02to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about0.08. The ratio of the starting materials (input) also falls within thisrange. In other embodiments, the particle preparation uses about 400 μgnucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratioof about 0.01 to about 0.08 and, more preferably, about 0.04, whichcorresponds to 1.25 mg of total lipid per 50 μg of nucleic acid. Inother preferred embodiments, the particle has a nucleic acid:lipid massratio of about 0.08.

In other embodiments, the lipid to nucleic acid ratios (mass/massratios) in a formed nucleic acid-lipid particle (e.g., SNALP) will rangefrom about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100(100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) toabout 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4(4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), fromabout 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1),from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25(25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) toabout 20 (20:1), from about 5 (5:1) to about 15 (15:1), from about 5(5:1) to about 10 (10:1), or about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9(9:1), 10 (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), 15 (15:1),16 (16:1), 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21 (21:1), 22(22:1), 23 (23:1), 24 (24:1), or 25 (25:1), or any fraction thereof orrange therein. The ratio of the starting materials (input) also fallswithin this range.

As previously discussed, the conjugated lipid may further include a CPL.A variety of general methods for making SNALP-CPLs (CPL-containingSNALP) are discussed herein. Two general techniques include the“post-insertion” technique, that is, insertion of a CPL into, forexample, a pre-formed SNALP, and the “standard” technique, wherein theCPL is included in the lipid mixture during, for example, the SNALPformation steps. The post-insertion technique results in SNALP havingCPLs mainly in the external face of the SNALP bilayer membrane, whereasstandard techniques provide SNALP having CPLs on both internal andexternal faces. The method is especially useful for vesicles made fromphospholipids (which can contain cholesterol) and also for vesiclescontaining PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of makingSNALP-CPLs are taught, for example, in U.S. Pat. Nos. 5,705,385;6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent PublicationNo. 20020072121; and PCT Publication No. WO 00/62813, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

VI. Kits

The present invention also provides lipid particles (e.g., SNALP) in kitform. In some embodiments, the kit comprises a container which iscompartmentalized for holding the various elements of the lipidparticles (e.g., the active agents or therapeutic agents such as nucleicacids and the individual lipid components of the particles). Preferably,the kit comprises a container (e.g., a vial or ampoule) which holds thelipid particles of the invention (e.g., SNALP), wherein the particlesare produced by one of the processes set forth herein. In certainembodiments, the kit may further comprise an endosomal membranedestabilizer (e.g., calcium ions). The kit typically contains theparticle compositions of the invention, either as a suspension in apharmaceutically acceptable carrier or in dehydrated form, withinstructions for their rehydration (if lyophilized) and administration.

As explained herein, it has surprisingly been found that the SNALPformulations of the present invention containing at least one cationiclipid of Formulas I-XIV, either alone or in combination with othercationic lipids, show increased potency and/or increased tolerabilitywhen targeting a gene of interest in the liver, such as, e.g., APOB,APOC3, PCSK9, DGAT1, and/or DGAT2, when compared to other SNALPformulations. For instance, as set forth in the Examples below, it hasbeen found that a lipid particle (e.g., SNALP) containing, e.g.,DLin-K-C2-DMA (“C2K”), γ-DLenDMA, Linoleyl/Linolenyl DMA (“Lin/Len”),C2-DPanDMA, DPan-C2K-DMA, DPan-C3K-DMA, γ-DLen-C2K-DMA, DLen-C2K-DMA, orC2-TLinDMA was unexpectedly more potent in silencing APOB expression invivo compared to SNALP containing DLinDMA or DLenDMA. In addition, asset forth in the Examples below, it has been found that a lipid particle(e.g., SNALP) comprising an APOB siRNA described herein and containing,e.g., DLin-K-C2-DMA, displayed an unexpectedly more favorable toxicityprofile in vivo compared to SNALP formulations containing DLinDMA. Assuch, in certain preferred embodiments, the kits of the inventioncomprise a 1:57, 1:62, 7:54, or 7:58 lipid particle (e.g., SNALP)containing one or more cationic lipids of Formulas I-XIV, such as C2K,γ-DLenDMA, Linoleyl/Linolenyl DMA (“Lin/Len”), C2-DPanDMA, DPan-C2K-DMA,DPan-C3K-DMA, γ-DLen-C2K-DMA, DLen-C2K-DMA, and/or C2-TLinDMA. Those ofskill in the art will appreciate that the lipid particles can be presentin a container as a suspension or in dehydrated form.

In certain other instances, it may be desirable to have a targetingmoiety attached to the surface of the lipid particle to further enhancethe targeting of the particle. Methods of attaching targeting moieties(e.g., antibodies, proteins, etc.) to lipids (such as those used in thepresent particles) are known to those of skill in the art.

VII. Administration of Lipid Particles

Once formed, the lipid particles of the invention (e.g., SNALP) areparticularly useful for introducing an interfering RNA (e.g., an siRNAmolecule) targeting a gene of interest (such as APOB, APOC3, PCSK9,DGAT1, DGAT2, or combinations thereof) into the liver. As noted, it hassurprisingly been found that the SNALP formulations of the presentinvention containing a cationic lipid of Formula I-XIV are unexpectedlymore potent at silencing APOB expression and/or display increasedtolerability in vivo compared to SNALP formulations containing othercationic lipids such as DLinDMA. Accordingly, the present invention alsoprovides methods for introducing an interfering RNA (e.g., an siRNA)into a liver cell. The methods are carried out in vitro or in vivo byfirst forming the particles as described above and then contacting theparticles with the cells (e.g., cells of the liver, such as hepatocytes)for a period of time sufficient for delivery of the interfering RNA tothe liver cells to occur.

The lipid particles of the invention (e.g., SNALP) can be adsorbed toalmost any cell type with which they are mixed or contacted. Onceadsorbed, the particles can either be endocytosed by a portion of thecells, exchange lipids with cell membranes, or fuse with the cells.Transfer or incorporation of the active agent or therapeutic agent(e.g., nucleic acid) portion of the particle can take place via any oneof these pathways. In particular, when fusion takes place, the particlemembrane is integrated into the cell membrane and the contents of theparticle combine with the intracellular fluid.

The lipid particles of the invention (e.g., SNALP) can be administeredeither alone or in a mixture with a pharmaceutically acceptable carrier(e.g., physiological saline or phosphate buffer) selected in accordancewith the route of administration and standard pharmaceutical practice.Generally, normal buffered saline (e.g., 135-150 mM NaCl) will beemployed as the pharmaceutically acceptable carrier. Other suitablecarriers include, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like, including glycoproteins for enhanced stability,such as albumin, lipoprotein, globulin, etc. Additional suitablecarriers are described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES,Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As usedherein, “carrier” includes any and all solvents, dispersion media,vehicles, coatings, diluents, antibacterial and antifungal agents,isotonic and absorption delaying agents, buffers, carrier solutions,suspensions, colloids, and the like. The phrase “pharmaceuticallyacceptable” refers to molecular entities and compositions that do notproduce an allergic or similar untoward reaction when administered to ahuman.

The pharmaceutically acceptable carrier is generally added followinglipid particle formation. Thus, after the lipid particle (e.g., SNALP)is formed, the particle can be diluted into pharmaceutically acceptablecarriers such as normal buffered saline.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2 to 5%, to as much as about 10 to 90% by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration.

The pharmaceutical compositions of the present invention may besterilized by conventional, well-known sterilization techniques. Aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, andcalcium chloride. Additionally, the particle suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol, and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

In some embodiments, the lipid particles of the invention (e.g., SNALP)are particularly useful in methods for the therapeutic delivery of oneor more nucleic acids comprising an interfering RNA sequence (e.g.,siRNA). In particular, it is an object of this invention to provide invitro and in vivo methods for treatment of a disease or disorder in amammal (e.g., a rodent such as a mouse or a primate such as a human,chimpanzee, or monkey) by downregulating or silencing the transcriptionand/or translation of one or more target nucleic acid sequences or genesof interest (such as APOB, APOC3, PCSK9, DGAT1, DGAT2, or combinationsthereof). As a non-limiting example, the methods of the invention areuseful for in vivo delivery of interfering RNA (e.g., siRNA) to theliver of a mammalian subject for the treatment of metabolic diseases anddisorders (e.g., diseases and disorders in which the liver is a targetand liver diseases and disorders). In certain embodiments, the diseaseor disorder is associated with expression and/or overexpression of agene and expression or overexpression of the gene is reduced by theinterfering RNA (e.g., siRNA). In certain other embodiments, atherapeutically effective amount of the lipid particle may beadministered to the mammal. In some instances, an interfering RNA (e.g.,siRNA) is formulated into a SNALP containing a cationic lipid of FormulaI-XIV, and the particles are administered to patients requiring suchtreatment. In other instances, cells are removed from a patient, theinterfering RNA is delivered in vitro (e.g., using a SNALP describedherein), and the cells are reinjected into the patient.

A. In vivo Administration

Systemic delivery for in vivo therapy, e.g., delivery of a therapeuticnucleic acid to a distal target cell via body systems such as thecirculation, has been achieved using nucleic acid-lipid particles suchas those described in PCT Publication Nos. WO 05/007196, WO 05/121348,WO 05/120152, and WO 04/002453, the disclosures of which are hereinincorporated by reference in their entirety for all purposes. Thepresent invention also provides fully encapsulated lipid particles thatprotect the nucleic acid from nuclease degradation in serum, arenon-immunogenic, are small in size, and are suitable for repeat dosing.

For in vivo administration, administration can be in any manner known inthe art, e.g., by injection, oral administration, inhalation (e.g.,intransal or intratracheal), transdermal application, or rectaladministration. Administration can be accomplished via single or divideddoses. The pharmaceutical compositions can be administered parenterally,i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In some embodiments, thepharmaceutical compositions are administered intravenously orintraperitoneally by a bolus injection (see, e.g., U.S. Pat. No.5,286,634). Intracellular nucleic acid delivery has also been discussedin Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino etal., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. DrugCarrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).Still other methods of administering lipid-based therapeutics aredescribed in, for example, U.S. Pat. Nos. 3,993,754; 4,145,410;4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles canbe administered by direct injection at the site of disease or byinjection at a site distal from the site of disease (see, e.g., Culver,HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71 (1994)). The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

In embodiments where the lipid particles of the present invention (e.g.,SNALP) are administered intravenously, at least about 5%, 10%, 15%, 20%,or 25% of the total injected dose of the particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In other embodiments,more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% ofthe total injected dose of the lipid particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In certain instances,more than about 10% of a plurality of the particles is present in theplasma of a mammal about 1 hour after administration. In certain otherinstances, the presence of the lipid particles is detectable at leastabout 1 hour after administration of the particle. In certainembodiments, the presence of a therapeutic agent such as a nucleic acidis detectable liver cells (e.g., hepatocytes) at about 8, 12, 24, 36,48, 60, 72 or 96 hours after administration. In other embodiments,downregulation of expression of a target sequence, such as APOB, by aninterfering RNA (e.g., siRNA) is detectable at about 8, 12, 24, 36, 48,60, 72 or 96 hours after administration. In yet other embodiments,downregulation of expression of a target sequence, such as APOB, by aninterfering RNA (e.g., siRNA) occurs preferentially in liver cells(e.g., hepatocytes). In further embodiments, the presence or effect ofan interfering RNA (e.g., siRNA) in cells at a site proximal or distalto the site of administration or in liver cells (e.g., hepatocytes) isdetectable at about 12, 24, 48, 72, or 96 hours, or at about 6, 8, 10,12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration. Inadditional embodiments, the lipid particles (e.g., SNALP) of theinvention are administered parenterally or intraperitoneally.

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering nucleic acid compositions directly tothe lungs via nasal aerosol sprays have been described, e.g., in U.S.Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs usingintranasal microparticle resins and lysophosphatidyl-glycerol compounds(U.S. Pat. No. 5,725,871) are also well-known in the pharmaceuticalarts. Similarly, transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045. The disclosures of the above-described patents are hereinincorporated by reference in their entirety for all purposes.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions are preferablyadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically, or intrathecally.

Generally, when administered intravenously, the lipid particleformulations are formulated with a suitable pharmaceutical carrier. Manypharmaceutically acceptable carriers may be employed in the compositionsand methods of the present invention. Suitable formulations for use inthe present invention are found, for example, in REMINGTON'SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). A variety of aqueous carriers may be used, for example,water, buffered water, 0.4% saline, 0.3% glycine, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Generally, normal buffered saline (135-150mM NaCl) will be employed as the pharmaceutically acceptable carrier,but other suitable carriers will suffice. These compositions can besterilized by conventional liposomal sterilization techniques, such asfiltration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. Thesecompositions can be sterilized using the techniques referred to aboveor, alternatively, they can be produced under sterile conditions. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles disclosed herein may bedelivered via oral administration to the individual. The particles maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, pills, lozenges, elixirs,mouthwash, suspensions, oral sprays, syrups, wafers, and the like (see,e.g., U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes). These oral dosage forms may also contain thefollowing: binders, gelatin; excipients, lubricants, and/or flavoringagents. When the unit dosage form is a capsule, it may contain, inaddition to the materials described above, a liquid carrier. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. Of course, any material used inpreparing any unit dosage form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed.

Typically, these oral formulations may contain at least about 0.1% ofthe lipid particles or more, although the percentage of the particlesmay, of course, be varied and may conveniently be between about 1% or 2%and about 60% or 70% or more of the weight or volume of the totalformulation. Naturally, the amount of particles in each therapeuticallyuseful composition may be prepared is such a way that a suitable dosagewill be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

Formulations suitable for oral administration can consist of: (a) liquidsolutions, such as an effective amount of a packaged therapeutic agentsuch as nucleic acid (e.g., interfering RNA) suspended in diluents suchas water, saline, or PEG 400; (b) capsules, sachets, or tablets, eachcontaining a predetermined amount of a therapeutic agent such as nucleicacid (e.g., interfering RNA), as liquids, solids, granules, or gelatin;(c) suspensions in an appropriate liquid; and (d) suitable emulsions.Tablet forms can include one or more of lactose, sucrose, mannitol,sorbitol, calcium phosphates, corn starch, potato starch,microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc,magnesium stearate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, moistening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically compatible carriers. Lozenge forms can comprise atherapeutic agent such as nucleic acid (e.g., interfering RNA) in aflavor, e.g., sucrose, as well as pastilles comprising the therapeuticagent in an inert base, such as gelatin and glycerin or sucrose andacacia emulsions, gels, and the like containing, in addition to thetherapeutic agent, carriers known in the art.

In another example of their use, lipid particles can be incorporatedinto a broad range of topical dosage forms. For instance, a suspensioncontaining nucleic acid-lipid particles such as SNALP can be formulatedand administered as gels, oils, emulsions, topical creams, pastes,ointments, lotions, foams, mousses, and the like.

When preparing pharmaceutical preparations of the lipid particles of theinvention, it is preferable to use quantities of the particles whichhave been purified to reduce or eliminate empty particles or particleswith therapeutic agents such as nucleic acid associated with theexternal surface.

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as primates(e.g., humans and chimpanzees as well as other nonhuman primates),canines, felines, equines, bovines, ovines, caprines, rodents (e.g.,rats and mice), lagomorphs, and swine.

The amount of particles administered will depend upon the ratio oftherapeutic agent (e.g., nucleic acid) to lipid, the particulartherapeutic agent (e.g., nucleic acid) used, the disease or disorderbeing treated, the age, weight, and condition of the patient, and thejudgment of the clinician, but will generally be between about 0.01 andabout 50 mg per kilogram of body weight, preferably between about 0.1and about 5 mg/kg of body weight, or about 10⁸-10¹⁰ particles peradministration (e.g., injection).

B. In vitro Administration

For in vitro applications, the delivery of therapeutic agents such asnucleic acids (e.g., interfering RNA) can be to any cell grown inculture, whether of plant or animal origin, vertebrate or invertebrate,and of any tissue or type. In preferred embodiments, the cells areanimal cells, more preferably mammalian cells, and most preferably humancells (e.g., liver cells, i.e., hepatocytes).

Contact between the cells and the lipid particles, when carried out invitro, takes place in a biologically compatible medium. Theconcentration of particles varies widely depending on the particularapplication, but is generally between about 1 μmol and about 10 mmol.Treatment of the cells with the lipid particles is generally carried outat physiological temperatures (about 37° C.) for periods of time of fromabout 1 to 48 hours, preferably of from about 2 to 4 hours.

In one group of preferred embodiments, a lipid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/ml, more preferably about 2×10⁴ cells/ml.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 0.2 μg/ml, more preferably about 0.1 μg/ml.

To the extent that tissue culture of cells may be required, it iswell-known in the art. For example, Freshney, Culture of Animal Cells, aManual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchleret al., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977), and the references cited thereinprovide a general guide to the culture of cells. Cultured cell systemsoften will be in the form of monolayers of cells, although cellsuspensions are also used.

Using an Endosomal Release Parameter (ERP) assay, the deliveryefficiency of the SNALP or other lipid particle of the invention can beoptimized. An ERP assay is described in detail in U.S. PatentPublication No. 20030077829, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Moreparticularly, the purpose of an ERP assay is to distinguish the effectof various cationic lipids and helper lipid components of SNALP or otherlipid particle based on their relative effect on binding/uptake orfusion with/destabilization of the endosomal membrane. This assay allowsone to determine quantitatively how each component of the SNALP or otherlipid particle affects delivery efficiency, thereby optimizing the SNALPor other lipid particle. Usually, an ERP assay measures expression of areporter protein (e.g., luciferase, β-galactosidase, green fluorescentprotein (GFP), etc.), and in some instances, a SNALP formulationoptimized for an expression plasmid will also be appropriate forencapsulating an interfering RNA. In other instances, an ERP assay canbe adapted to measure downregulation of transcription or translation ofa target sequence in the presence or absence of an interfering RNA(e.g., siRNA). By comparing the ERPs for each of the various SNALP orother lipid particles, one can readily determine the optimized system,e.g., the SNALP or other lipid particle that has the greatest uptake inthe cell.

C. Cells for Delivery of Lipid Particles

The compositions and methods of the present invention are particularlywell suited for treating metabolic diseases and disorders by targeting,e.g., APOB in vivo. In preferred embodiments, an interfering RNA (e.g.,an siRNA) in a SNALP formulation containing a cationic lipid of FormulaI-XIV is delivered to liver cells (e.g., hepatocytes), whichsurprisingly results in increased silencing of the target gene ofinterest (e.g., APOB). The methods and compositions can be employed withliver cells (e.g., hepatocytes) of a wide variety of vertebrates,including mammals, such as, e.g, canines, felines, equines, bovines,ovines, caprines, rodents (e.g., mice, rats, and guinea pigs),lagomorphs, swine, and primates (e.g. monkeys, chimpanzees, and humans).

D. Detection of Lipid Particles

In some embodiments, the lipid particles of the present invention (e.g.,SNALP) are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7, 8 ormore hours. In other embodiments, the lipid particles of the presentinvention (e.g., SNALP) are detectable in the subject at about 8, 12,24, 48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22,24, 25, or 28 days after administration of the particles. The presenceof the particles can be detected in the cells, tissues, or otherbiological samples from the subject. The particles may be detected,e.g., by direct detection of the particles, detection of a therapeuticnucleic acid such as an interfering RNA (e.g., siRNA) sequence,detection of the target sequence of interest (i.e., by detectingexpression or reduced expression of the sequence of interest), or acombination thereof.

1. Detection of Particles

Lipid particles of the invention such as SNALP can be detected using anymethod known in the art. For example, a label can be coupled directly orindirectly to a component of the lipid particle using methods well-knownin the art. A wide variety of labels can be used, with the choice oflabel depending on sensitivity required, ease of conjugation with thelipid particle component, stability requirements, and availableinstrumentation and disposal provisions. Suitable labels include, butare not limited to, spectral labels such as fluorescent dyes (e.g.,fluorescein and derivatives, such as fluorescein isothiocyanate (FITC)and Oregon Green™; rhodamine and derivatives such Texas red,tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin,phycoerythrin, AMCA, CyDyes™, and the like; radiolabels such as ³H,¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.; enzymes such as horse radish peroxidase,alkaline phosphatase, etc.; spectral colorimetric labels such ascolloidal gold or colored glass or plastic beads such as polystyrene,polypropylene, latex, etc. The label can be detected using any meansknown in the art.

2. Detection of Nucleic Acids

Nucleic acids (e.g., interfering RNA) are detected and quantified hereinby any of a number of means well-known to those of skill in the art. Thedetection of nucleic acids may proceed by well-known methods such asSouthern analysis, Northern analysis, gel electrophoresis, PCR,radiolabeling, scintillation counting, and affinity chromatography.Additional analytic biochemical methods such as spectrophotometry,radiography, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), and hyperdiffusion chromatography may also be employed.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in, e.g., “Nucleic Acid Hybridization, A PracticalApproach,” Eds. Hames and Higgins, IRL Press (1985).

The sensitivity of the hybridization assays may be enhanced through theuse of a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification method's, including the polymerase chain reaction (PCR),the ligase chain reaction (LCR), Qβ-replicase amplification, and otherRNA polymerase mediated techniques (e.g., NASBA™) are found in Sambrooket al., In Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2000); and Ausubel et al., SHORT PROTOCOLS INMOLECULAR BIOLOGY, eds., Current Protocols, Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc. (2002); as well as U.S.Pat. No. 4,683,202; PCR Protocols, A Guide to Methods and Applications(Innis et al. eds.) Academic Press Inc. San Diego, Calif. (1990);Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research,3:81 (1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989);Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell etal., J. Clin. Chem., 35:1826 (1989); Landegren et al., Science, 241:1077(1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene,4:560 (1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan andMalek, Biotechnology, 13:563 (1995). Improved methods of cloning invitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039.Other methods described in the art are the nucleic acid sequence basedamplification (NASBA™, Cangene, Mississauga, Ontario) and Qβ-replicasesystems. These systems can be used to directly identify mutants wherethe PCR or LCR primers are designed to be extended or ligated only whena select sequence is present. Alternatively, the select sequences can begenerally amplified using, for example, nonspecific PCR primers and theamplified target region later probed for a specific sequence indicativeof a mutation. The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

Nucleic acids for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as inhibitor components aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage et al.,Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an automatedsynthesizer, as described in Needham VanDevanter et al., Nucleic AcidsRes., 12:6159 (1984). Purification of polynucleotides, where necessary,is typically performed by either native acrylamide gel electrophoresisor by anion exchange HPLC as described in Pearson et al., J. Chrom.,255:137 149 (1983). The sequence of the synthetic polynucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology, 65:499.

An alternative means for determining the level of transcription is insitu hybridization. In situ hybridization assays are well-known and aregenerally described in Angerer et al., Methods Enzymol., 152:649 (1987).In an in situ hybridization assay, cells are fixed to a solid support,typically a glass slide. If DNA is to be probed, the cells are denaturedwith heat or alkali. The cells are then contacted with a hybridizationsolution at a moderate temperature to permit annealing of specificprobes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters.

VIII. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1 Synthesis of2,2-Dilinoleyl-4-Dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA)

DLin-K-DMA was synthesized as shown in the schematic and describedbelow.

Synthesis of Linoleyl Bromide (II)

A mixture of linoleyl methane sulfonate (6.2 g, 18 mmol) and magnesiumbromide etherate (17 g, 55 mmol) in anhydrous ether (300 mL) was stirredunder argon overnight (21 hours). The resulting suspension was pouredinto 300 mL of chilled water. Upon shaking, the organic phase wasseparated. The aqueous phase was extracted with ether (2×150 mL). Thecombined ether phase was washed with water (2×150 mL), brine (150 mL),and dried over anhydrous Na₂SO₄. The solvent was evaporated to afford6.5 g of colourless oil. The crude product was purified by columnchromatography on silica gel (230-400 mesh, 300 mL) and eluted withhexanes. This gave 6.2 g (approximately 100%) of linoleyl bromide (II).¹H NMR (400 MHz, CDCl₃) δ: 5.27-5.45 (4H, m, 2×CH═CH), 3.42 (2H, t,CH₂Br), 2.79 (2H, t, C═C—CH₂—C═C), 2.06 (4H, q, 2× allylic CH₂), 1.87(2H, quintet, CH₂), 1.2-1.5 (16H, m), 0.90 (3H, t, CH₃) ppm.

Synthesis of Dilinoleyl Methanol (III)

To a suspension of Mg turnings (0.45 g, 18.7 mmol) with one crystal ofiodine in 200 mL of anhydrous ether under nitrogen was added a solutionof linoleyl bromide (II) in 50 mL of anhydrous ether at roomtemperature. The resulting mixture was refluxed under nitrogenovernight. The mixture was cooled to room temperature. To the cloudymixture under nitrogen was added dropwise at room temperature a solutionof ethyl formate (0.65 g, 18.7 mmol) in 30 mL of anhydrous ether. Uponaddition, the mixture was stirred at room temperature overnight (20hours). The ether layer was washed with 10% H₂SO₄ aqueous solution (100mL), water (2×100 mL), brine (150 mL), and then dried over anhydrousNa₂SO₄. Evaporation of the solvent gave 5.0 g of pale oil. Columnchromatography on silica gel (230-400 mesh, 300 mL) with 0-7% ethergradient in hexanes as eluent afforded two products, dilinoleyl methanol(2.0 g, III) and dilinoleylmethyl formate (1.4 g, IV). ¹H NMR (400 MHz,CDCl₃) for dilinoleylmethyl formate (IV) δ: 8.10 (1H, s, CHO), 5.27-5.45(8H, m, 4×CH═CH), 4.99 (1′-1, quintet, OCH), 2.78 (4H, t,2×C═C—CH₂—C═C), 2.06 (8H, q, 4× allylic CH₂), 1.5-1.6 (4H, m, 2×CH₂),1.2-1.5 (32H, m), 0.90 (6H, t, 2×CH₃) ppm.

Dilinoleylmethyl formate (IV, 1.4 g) and KOH (0.2 g) were stirred in 85%EtOH at room temperature under nitrogen overnight. Upon completion ofthe reaction, half of the solvent was evaporated. The resulting mixturewas poured into 150 mL of 5% HCL solution. The aqueous phase wasextracted with ether (3×100 mL). The combined ether extract was washedwith water (2×100 mL), brine (100 mL), and dried over anhydrous Na₂SO₄.Evaporation of the solvent gave 1.0 g of dilinoleyl methanol (III) ascolourless oil. Overall, 3.0 g (60%) of dilinoleyl methanol (III) wereafforded. ¹H NMR (400 MHz, CDCl₃) for dilinoleyl methanol (III) δ: ppm.

Synthesis of Dilinoleyl Ketone (V)

To a mixture of dilinoleyl methanol (2.0 g, 3.8 mmol) and anhydroussodium carbonate (0.2 g) in 100 mL of CH₂Cl₂ was added pydimiumchlorochromate (PCC, 2.0 g, 9.5 mmol). The resulting suspension wasstirred at room temperature for 60 min. Ether (300 mL) was then addedinto the mixture, and the resulting brown suspension was filteredthrough a pad of silica gel (300 mL). The silica gel pad was furtherwashed with ether (3×200 mL). The ether filtrate and washes werecombined. Evaporation of the solvent gave 3.0 g of an oily residual as acrude product. The crude product was purified by column chromatographyon silica gel (230-400 mesh, 250 mL) eluted with 0-3% ether in hexanes.This gave 1.8 g (90%) of dilinoleyl ketone (V). ¹H NMR (400 MHz, CDCl₃)δ: 5.25-5.45 (8H, m, 4×CH═CH), 2.78 (4H, t, 2×C═C—CH₂—C═C), 2.39 (4H, t,2×COCH₂), 2.05 (8H, q, 4× allylic CH₂), 1.45-1.7 (4H, m), 1.2-1.45 (32H,m), 0.90 (6H, t, 2×C₁₋₁₃) ppm.

Synthesis of 2,2-Dilinoleyl-4-bromomethyl-[1,3]-dioxolane (VI)

A mixture of dilinoleyl methanol (V, 1.3 g, 2.5 mmol),3-bromo-1,2-propanediol (1.5 g, 9.7 mmol) and p-toluene sulonic acidhydrate (0.16 g, 0.84 mmol) in 200 mL of toluene was refluxed undernitrogen for 3 days with a Dean-Stark tube to remove water. Theresulting mixture was cooled to room temperature. The organic phase waswashed with water (2×50 mL), brine (50 mL), and dried over anhydrousNa₂SO₄. Evaporation of the solvent resulted in a yellowish oily residue.Column chromatography on silica gel (230-400 mesh, 100 mL) with 0-6%ether gradient in hexanes as eluent afforded 0.1 g of pure VI and 1.3 gof a mixture of VI and the starting material. ¹H NMR (400 MHz, CDCl₃) δ:5.27-5.45 (8H, m, 4×CH═CH), 4.28-4.38 (1H, m, OCH), 4.15 (1H, dd, OCH),3.80 (1H, dd, OCH), 3.47 (1H, dd, CHBr), 3.30 (1H, dd, CHBr), 2.78 (4H,t, 2×C═C—CH₂—C═C), 2.06 (8H, q, 4× allylic CH₂), 1.52-1.68 (4H, m,2×CH₂), 1.22-1.45 (32H, m), 0.86-0.94 (6H, m, 2×CH₃) ppm.

Synthesis of 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA)

Anhydrous dimethyl amine was bubbled into an anhydrous THF solution (100mL) containing 1.3 g of a mixture of2,2-dilinoleyl-4-bromomethyl-[1,3]-dioxolane (VI) and dilinoleyl ketone(V) at 0° C. for 10 min. The reaction flask was then sealed and themixture stirred at room temperature for 6 days. Evaporation of thesolvent left 1.5 g of a residual. The crude product was purified bycolumn chromatography on silica gel (230-400 mesh, 100 mL) and elutedwith 0-5% methanol gradient in dichloromethane. This gave 0.8 g of thedesired product DLin-K-DMA. ¹H NMR (400 MHz, CDCl₃) δ: 5.25-5.45 (8, m,4×CH═CH), 4.28-4.4 (1H, m, OCH), 4.1 (1H, dd, OCH), 3.53 (1H, t OCH),2.78 (4H, t, 2×C═C—CH₂—C═C), 2.5-2.65 (2H, m, NCH₂), 2.41 (6H, s,2×NCH₃), 2.06 (8H, q, 4× allylic CH₂), 1.56-1.68 (4H, m, 2×CH₂),1.22-1.45 (32H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 2 Synthesis of2,2-Dilinoleyl-4-(2-Dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA)

DLin-K-C2-DMA was synthesized as shown in the schematic diagram anddescription below.

Synthesis of 2,2-Dilinoleyl-4-(2-hydroxyethyl)[1,3]-dioxolane (II)

A mixture of dilinoleyl ketone (I, previously prepared as described inExample 1, 527 mg, 1.0 mmol), 1,3,4-butanetriol (technical grade, ca.90%, 236 mg, 2 mmol) and pyridinium p-toluenesulfonate (50 mg, 0.2 mmol)in 50 mL of toluene was refluxed under nitrogen overnight with aDean-Stark tube to remove water. The resulting mixture was cooled toroom temperature. The organic phase was washed with water (2×30 mL),brine (50 mL), and dried over anhydrous Na₂SO₄. Evaporation of thesolvent resulted in a yellowish oily residual (0.6 g). The crude productwas purified by column chromatography on silica gel (230-400 mesh, 100mL) with dichloromethane as eluent. This afforded 0.5 g of pure II ascolourless oil. ¹H NMR (400 MHz, CDCl₃) δ: 5.25-5.48 (8H, m, 4×CH═CH),4.18-4.22 (1H, m, OCH), 4.08 (1H, dd, OCH), 3.82 (2H, t, OCH₂), 3.53(1H, t, OCH), 2.78 (4H, t, 2×C═C—CH₂—C═C), 2.06 (8H, q, 4× allylic CH₂),1.77-1.93 (2H, m, CH₂), 1.52-1.68 (4H, m, 2×CH₂), 1.22-1.45 (32H, m),0.86-0.94 (6H, t, 2×CH₃) ppm.

Synthesis of 2,2-Dilinoleyl-4-(2-methanesulfonylethyl)-[1,3]-dioxolane(III)

To a solution of 2,2-dilinoleyl-4-(2-hydroxyethyl)-[1,3]-dioxolane (II,500 mg, 0.81 mmol) and dry triethylamine (218 mg, 2.8 mmol) in 50 mL ofanhydrous CH₂Cl₂ was added methanesulfonyl anhydride (290 mg, 1.6 mmol)under nitrogen. The resulting mixture was stirred at room temperatureovernight. The mixture was diluted with 25 mL of CH₂Cl₂. The organicphase was washed with water (2×30 mL), brine (50 mL), and dried overanhydrous Na₂SO₄. The solvent was evaporated to afford 510 mg ofyellowish oil. The crude product was used in the following step withoutfurther purification.

Synthesis of 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane(DLin-K-C2-DMA)

To the above crude material (III) under nitrogen was added 20 mL ofdimethylamine in THF (2.0 M). The resulting mixture was stirred at roomtemperature for 6 days. An oily residual was obtained upon evaporationof the solvent. Column chromatography on silica gel (230-400 mesh, 100mL) with 0-5% methanol gradient in dichloromethane as eluent resulted in380 mg of the product DLin-K-C2-DMA as pale oil. ¹H NMR (400 MHz, CDCl₃)δ: 5.27-5.49 (8, m, 4×CH═CH), 4.01-4.15 (2H, m, 2×OCH), 3.49 (1H, tOCH), 2.78 (4H, t, 2×C═C—CH₂—C═C), 2.34-2.54 (2H, m, NCH₂), 2.30 (6H, s,2×NCH₃), 2.06 (8H, q, 4× allylic CH₂), 1.67-1.95 (2H, m, CH₂), 1.54-1.65(4H, m, 2×CH₂), 1.22-1.45 (32H, m), 0.90 (6H, t, 2×CH₃) ppm.

Example 3 Synthesis of 1,2-Di-γ-linolenyloxy-N,N-dimethylaminopropane(γ-DLenDMA)

γ-DLenDMA having the structure shown below was synthesized as describedbelow.

A 250 mL round bottom flask was charged with3-(dimethylamino)-1,2-propanediol (0.8 g, 6.7 mmol), tetrabutylammoniumhydrogen sulphate (1 g), gamma linolenyl mesylate(cis-6,9,12-octadecatriene sulphonic acid) (5 g, 14.6 mmol), and 30 mLtoluene. After stirring for 15 minutes, the reaction was cooled to 0-5°C. A solution of 40% sodium hydroxide (15 mL) was added slowly. Thereaction was left to stir for approximately 48 hours. An additional 15mL of toluene was then added to the reaction vessel, along with 40%sodium hydroxide (15 mL). After the reaction was stirred for anadditional 12 hours, water (50 mL) and isopropyl acetate (50 mL) wereadded and stirred for 15 minutes. The mixture was then transferred to a500 mL separatory funnel and allowed to separate. The lower aqueousphase was run off and the organic phase was washed with saturated sodiumchloride (2×50 mL). Since the aqueous and organic phases resulting fromthe saturated sodium chloride washes could not be completely separatedafter 20 minutes, the lower aqueous phase (slightly yellow) was run offand back extracted with chloroform (˜45 mL). The organic phase was driedwith MgSO₄, filtered, and the solvent evaporated.

The crude product, an orange liquid, was purified on columnchromatography using silica gel (60 g) with 0-3% methanol gradient indichloromethane to yield 3.19 g. The product was further purified viacolumn chromatography on silica gel (50 g) with 10-30% ethyl acetategradient in hexanes to yield 1.26 g pure product.

Example 4 Synthesis of 1,2-Diphytanyloxy-3-(N,N-dimethyl)-propylamine(DPanDMA)

DPanDMA having the structure shown below was synthesized as describedbelow.

Step 1: Synthesis of Phytanol:

Phytol (21.0 g, 70.8 mmol), ethanol (180 mL) and a stir bar were addedto a 500 mL round bottom flask. Raney Nickel 2800 (as purchased, a 50%by weight solution in water if used as purchased, Nickel >89% metalpresent) (6.8 g, 51.5 mmol) was added, and the flask sealed and flushedwith hydrogen. A 12″ needle was used to bubble hydrogen through thesolution for 10 minutes. The reaction was stirred for 5 days, using aballoon as a hydrogen reservoir. Hydrogen was also bubbled through thereaction mixture at 24 h and 48 h, 5 minutes each time. The metalcatalyst was then removed by filtering through Celite. The ethanolicsolution was concentrated, and 200 mL of DCM added to the resulting oil.The solution was washed with water (2×100 mL), dried over MgSO₄, andconcentrated. TLC indicated formation of the phytanol product, yield20.0 g.

Step 2: Synthesis of Phytanyl Mesylate:

Phytanyl (20.0 g, 66.7 mmol), triethylamine (18.6 mL, 133 mmol), and astir bar were added to a 1000 mL round bottom flask. The flask wassealed and flushed with nitrogen. Anhydrous DCM (250 mL) was added, andthe mixture cooled to −15° C. (ice and NaCl). Mesyl Chloride (10.4 mL,133 mmol) was added slowly via syringe over a 30 minute period, and thereaction stirred at −15° C. for a further 1.5 hours. At this point TLCshowed that the starting material had been used up. The solution wasdiluted with DCM (250 mL) and washed with saturated NaHCO₃ (2×200 mL).The organic phase was then dried (MgSO₄), filtered, and concentrated(rotovap). The crude product was purified by column chromatography.Yield: 21.5 g, 85.7%.

Step 3: Synthesis of DPanDMA:

Sodium hydride (2.5 g, 100 mmol) was added to a 250 mL round bottomflask, along with benzene (40 mL) and a stir bar. In a 50 mL beaker, asolution was made from the N,N-Dimethyl-3-aminopropane-1,2-diol (1.42 g,12 mmol) and benzene (60 mL). This was added to the reaction vessel andthe reaction stirred for 10 minutes (effervescence). Phytanyl Mesylate(10.52 g, 28 mmol) was added and the flask fitted with a condenser,flushed with nitrogen, and heated to reflux. After 18 hours, the flaskwas removed from the heat and allowed to cool. The volume was made up to200 mL with benzene. EtOH was added slowly to quench unreacted sodiumhydride. Once quenching was complete, the reaction mixture was washedtwice with EtOH/H₂O, in a ratio to the benzene of 1:1:0.6benzene:water:ethanol. The aqueous phases were combined and extractedwith CHCl₃ (2×100 mL). Finally, the organic phase was dried (MgSO₄),filtered, and concentrated (rotovap). Purification by columnchromatography yielded DPanDMA as a pale yellow oil (6.1 g, 8.97 mmol,74.7%).

Example 5 Synthesis of Cationic Lipids of the TLinDMA Family

The following diagram provides a general scheme for synthesizing membersof the C(n)-TLinDMA family of cationic lipids:

TLinDMA(1-(2,3-linoleyloxypropoxy)-2-(linoleyloxy)-(N,N-dimethyl)-propyl-3-amine)(Compound III) was synthesized as follows:

Synthesis of Compound I:

A 1000 ml round bottom flask was charged with epibromohydrin (5 g, 37mmol), glycerol (10 g, 110 mmol), a stir bar and then flushed withnitrogen. Anhydrous chloroform (350 mL) was added via cannula, followedby BF₃.Et₂O (0.5 mL, 3.7 mmol) and refluxed for 3 hours under nitrogen.The reaction mixture was cooled and subsequently stirred at roomtemperature overnight. Upon completion of the reaction, the reactionmixture was concentrated and the crude product (15 g) was purified viacolumn chromatography using silica gel (150 g).

Synthesis of Compound II:

A 500 mL round bottom flask was charged with Compound I (3.8 g, 17 mmol)and a stir bar. After flushing with nitrogen, dimethylamine in a 2.0 Mmethyl alcohol solution (170 mL) was added via cannula. The resultingmixture was stirred at room temperature for 48 hours. The progress ofthe reaction was monitored using TLC. The crude product was used withoutfurther purification.

Synthesis of TLinDMA (Compound III):

A 100 mL round bottom flask was charged with a stir bar, NaH (0.6 g, 24mmol), and 25 mL benzene. Subsequently, Compound II (0.4 g, 2 mmol) wasadded followed immediately by linoleyl methane sulfonate (2.8 g, 8mmol). The reaction was flushed with nitrogen and refluxed overnight.Progress of the reaction was monitored via TLC. The reaction mixture wastransferred to a 250 mL separatory funnel and diluted with benzene to afinal volume of 50 mL. The reaction was quenched with ethanol (30 mL)and then washed with water (50 mL). The lower aqueous phase was run offand the reaction mixture was washed again with ethanol (30 mL) and water(50 mL). The organic phase was dried with MgSO₄, filtered, and solventremoved. The crude product (2.3 g) was purified via columnchromatography on silica gel (60 g) with 0-3% methanol gradient indichloromethane.

C2-TLinDMA (Compound VII) was synthesized as follows:

Synthesis of Compound IV:

A solution of 4-bromo-1-butene (11.5 g, 85 mmol) in CH₂Cl₂ (anh., 120ml) was prepared under nitrogen in a 1000 ml RBF with a magneticstirrer. In a separate flask, a solution of 3-chloroperbenzoic acid(77%, MW 173, 44.05 g, 196 mmol) in CH₂Cl₂ (anh., 250 ml) prepared andadded to the reaction mixture by canulla. The reaction was stirred for 3days, and then concentrated. The product (oil/white solid mixture) wasre-dissolved in THF (300 mL) and a solution of 4% sodium dithionite (180mL) added to remove excess peracid. The mixture (now cloudy) was stirredfor 20 minutes and then EtOAc (750 mL) added. The mixture wastransferred to a separating funnel and the organic was washed with water(100 mL), sat. NaHCO₃ (2×300 mL, EFFERVESCENCE), water again (300 mL)and brine (300 mL). The solution was concentrated and the productpurified by chromatography.

Synthesis of Compound V:

A 250 ml round bottom flask was charged with Compound IV (1.3 g, 9mmol), glycerol (2.5 g, 27 mmol), a stir bar and then flushed withnitrogen. Anhydrous chloroform (100 mL) was added via cannula, followedby BF₃.Et₂O (0.15 mL, 1.1 mmol) and refluxed for 3 hours under nitrogen.The reaction mixture was subsequently stirred at room temperatureovernight.

Synthesis of Compound VI:

A 50 mL round bottom flask was charged with Compound V (0.3 g, 1.2 mmol)and a stir bar. After flushing with nitrogen, dimethylamine in a 2.0 Mmethyl alcohol solution (25 mL) was added via syringe. The resultingmixture was stirred at room temperature for 48 hours. The progress ofthe reaction was monitored using t.l.c. The reaction mixture wasconcentrated and the crude product used without further purification.

Synthesis of C2-TLinDMA (Compound VII):

A 100 mL round bottom flask was charged with a stir bar, NaH (0.6 g, 24mmol), and 25 mL benzene. Compound VI (0.37 g, 1.8 mmol) was addedfollowed immediately by linoleyl methane sulfonate (2.8 g, 8 mmol). Thereaction was refluxed overnight and progress of the reaction wasmonitored via t.l.c. The reaction mixture was transferred to a 250 mLseparatory funnel and diluted with benzene to a final volume of 50 mL.The reaction was quenched with ethanol (30 mL) and then washed withwater (50 mL). The lower aqueous phase was run off and the reactionmixture washed again with ethanol (30 mL) and water (50 mL). The organicphase was dried with MgSO₄, filtered, and solvent removed. The crudeproduct, 2.5 g, was purified using column chromatography on silica gel(60 g), eluted with 0-3% methanol gradient in DCM.

C3-TLinDMA (Compound XI) was synthesized as follows:

Synthesis of Compound VIII:

A solution of 5-bromo-1-pentene (85 mmol) in CH₂Cl₂ (anh., 120 ml) isprepared under nitrogen in a 1000 ml RBF with a magnetic stirrer. In aseparate flask, a solution of 3-chloroperbenzoic acid (77%, MW 173,44.05 g, 196 mmol) in CH₂Cl₂ (anh., 250 ml) is prepared and added to thereaction mixture by canulla. The reaction is stirred for 3 days, andthen concentrated. The product (oil/white solid mixture) is re-dissolvedin THF (300 mL) and a solution of 4% sodium dithionite (180 mL) added toremove excess peracid. The mixture (now cloudy) is stirred for 20minutes and then EtOAc (750 mL) added. The mixture is transferred to aseparating funnel and the organic is washed with water (100 mL), sat.NaHCO₃ (2×300 mL, EFFERVESCENCE), water again (300 mL) and brine (300mL). The solution is concentrated and the product purified bychromatography.

Synthesis of Compound IX:

A 250 ml round bottom flask is charged with Compound VIII (1.3 g, 9mmol), glycerol (2.5 g, 27 mmol), a stir bar and then flushed withnitrogen. Anhydrous chloroform (100 mL) is added via cannula, followedby BF₃.Et₂O (0.15 mL, 1.1 mmol) and refluxed for 3 hours under nitrogen.The reaction mixture is subsequently stirred at room temperatureovernight.

Synthesis of Compound X:

A 50 mL round bottom flask is charged with Compound IX (0.3 g, 1.2 mmol)and a stir bar. After flushing with nitrogen, dimethylamine in a 2.0 Mmethyl alcohol solution (25 mL) is added via syringe. The resultingmixture is stirred at room temperature for 48 hours. The progress of thereaction is monitored using t.l.c. The reaction mixture is concentratedand the crude product used without further purification.

Synthesis of Compound XI:

A 100 mL round bottom flask is charged with a stir bar, NaH (0.6 g, 24mmol), and 25 mL benzene. Compound X (0.37 g, 1.8 mmol) is addedfollowed immediately by linoleyl methane sulfonate (2.8 g, 8 mmol). Thereaction is refluxed overnight and progress of the reaction monitoredvia t.l.c. The reaction mixture is transferred to a 250 mL separatoryfunnel and diluted with benzene to a final volume of 50 mL. The reactionis quenched with ethanol (30 mL) and then washed with water (50 mL). Thelower aqueous phase is run off and the reaction mixture washed againwith ethanol (30 mL) and water (50 mL). The organic phase is dried withMgSO₄, filtered, and solvent removed. The crude product, 2.5 g, ispurified using column chromatography on silica gel (60 g), eluted with0-3% methanol gradient in DCM.

Example 6 Synthesis of Novel C2 Lipids

Novel C2 lipids (Compounds V-VII) having the structures shown below weresynthesized as shown in the following schematic diagram.

Step 1: Synthesis of4-(2-Methanesulfonylethyl)-2,2-dimethyl-1,3-dioxolane (Compound I)

4-(2-Hydroxylethyl)-2,2-dimethyl-1,3-dioxolane (25 g, 170 mmol),triethylamine (55.9 mL, 400 mmol), and a stir bar were added to a 1000mL round bottom flask. The flask was sealed and flushed with nitrogen.Anhydrous DCM (600 mL) was added, and the mixture cooled to approx −5°C. (ice and NaCl). Mesyl chloride (19.9 mL, 255 mmol, 1.5 eq) was addedslowly via syringe over a 60 minute period, and the reaction stirred at−5° C. for a further 1.5 hours. At this point TLC showed that thestarting material had been consumed. The solution was diluted with DCM(350 mL), divided into two (˜500 mL) portions, and each portion workedup as follows: the solution was transferred to a 1000-mL separatingfunnel and washed with saturated NaHCO₃ (2×200 mL). The organic phasewas then dried (MgSO₄), filtered, and concentrated (rotovap). The crudeproduct was purified by column chromatography. Final yield: 32.0 g,84.1%.

Step 2: Synthesis of 4-(2-Bromoethyl)-2,2-dimethyl-1,3-dioxolane(Compound II)

Magnesium bromide etherate (40 g, 130 mmol) and a stir bar were added toa 2000 mL round bottom flask and flushed with nitrogen. A solution of4-(2-methanesulfonylethyl)-2,2-dimethyl-1,3-dioxolane (I) (17.5 g, 78mmol) in anhydrous diethyl ether (900 mL) was added via canulla, and thesuspension stirred overnight. The ether was first decanted into abeaker. Water (200 mL) and ether (300 mL) were added to the precipitateand stirred for 5 minutes. The precipitate was dissolved, and the etherphase was then collected and added to the ether solution from thereaction. The organic phase was then washed, concentrated to about 500mL, washed with water, dried over anhydrous Mg₂SO₄, filtered, andconcentrated to yield a yellow oil (16.0 g). This was purified by flashchromatography to yield 10.6 g of product (50.7 mmol, 65%).

Step 3: Synthesis of 4-Bromobutane-1,2-diol (Compound III)

4-(2-Bromoethyl)-2,2-dimethyl-1,3-dioxolane (II) (9 g, 43 mmol) wasadded to a 500 mL RBF with a stirbar. 100 mL of MeOH:H₂O:HCl in a ratioof (60:20:5) were added. After 30 minutes, sat. NaHCO₃ (˜75 mL) wasadded (effervescence), until pH paper indicated solution was basic. Atthis point the mixture was slightly cloudy. Ether (300 mL) was added(while stirring) and the cloudiness disappeared. The reaction mixturewas transferred to a 1000 mL sep funnel and the 2 phases separated. Theextraction of the aqueous phase was repeated two more times (2×300 mLether). Organics were combined, dried over MgSO₄ and concentrated toyield a colorless oil (7.0 g), which was purified by columnchromatography.

Step 4: Synthesis of 4-(Dimethylamino)-1,2-butanediol (Compound IV)

4-Bromobutane-1,2-diol (III) (1 g, 6.0 mmol) was added to a 50 mL RBFwith a stir bar, sealed, and flushed with nitrogen. 30 mL ofDimethylamine (2.0M solution in MeOH) was delivered by canulla and thereaction stirred overnight. TLC indicated all the starting material haddisappeared. The solvent (and DMA) were removed by evaporation and thecrude product used without further purification.

Synthesis of 1,2-Dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDMA)(Compound V)

4-(Dimethylamino)-1,2-butanediol (IV) (1.3 g, 3.4 mmol), linoleylmesylate (2.0 g, 5.8 mmol), tetrabutylammonium hydrogen sulphate (0.5 g,1.5 mmol), toluene (30 mL), and a stir bar were added to a 100 mL RBF.30 mL of 40% NaOH was made and added to the reaction mixture. Theresulting mixture was stirred at room temperature, under nitrogen for 60hours. Deionized water (50 mL) and isopropyl acetate (50 mL) were addedand the mixture stirred vigorously for a further 10-15 min. The mixturewas transferred to a 250 mL separating funnel and allowed to separateand the aqueous phase removed. The organic phase was washed twice withwater (2×30 mL) using MeOH to aid the separation, and the organic phasewas dried (MgSO₄), filtered, and concentrated to obtain a dark yellowoil. The oil was purified by column chromatography.

Synthesis of 1,2-Diphytanyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DPanDMA)(Compound VI)

Sodium hydride (360 mg, 15 mmol), benzene (40 mL), and a stir bar wereadded to a 50 mL round bottom flask. 4-(Dimethylamino)-1,2-butanediol(IV) (200 mg, 1.5 mmol) was added and the reaction stirred for 10minutes (effervescence). Phytanyl Mesylate (1.07 g, 2.92 mmol) was thenadded and the flask fitted with a condenser, flushed with nitrogen, andheated to reflux. After 18 hours, the flask was allowed to cool to roomtemperature. The volume was made up to 40 mL with benzene. EtOH wasadded slowly to quench unreacted sodium hydride. Once quenching wascomplete, the reaction mixture was washed twice with an EtOH/H₂O, in aratio to the benzene of 1:1:0.6 benzene:water:ethanol. The aqueouswashes were combined and extracted with CHCl₃ (2×20 mL). Finally, theorganics were combined, dried (MgSO₄), filtered, and concentrated(rotovap). Purification by column chromatography yielded a pale yellowoil (250 mg, 0.145 mmol, 25%).

Synthesis of 1,2-Dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine(C2-DLinDAP) (Compound VII)

A flask containing 4-(Dimethylamino)-1,2-butanediol (IV) (crude, 266 mg,2 mmol (max)), TEA (0.84 mL, 6 mmol), and DMAP (24 mg, 0.2 mmol) wasflushed with nitrogen before the addition of anhydrous CH₂Cl₂ (50 ml).Linoleoyl chloride (1.2 g, 4 mmol) was added and the solution stirredovernight. The solution was rinsed into a 250 mL separatory funnel withDCM (˜70 mL) and washed with water (2×50 mL). The organic was dried(MgSO₄), concentrated, and purified by chromatography.

Example 7 Synthesis of Novel Phytanyl Cationic Lipids

DPan-C2K-DMA, DPan-C1K6-DMA, and DPan-C3K-DMA having the structuresshown below were synthesized as shown in the following schematicdiagram.

Synthesis of Phytanol

Phytol (21.0 g, 70.8 mmol), ethanol (180 mL) and a stir bar were addedto a 500 mL round bottom flask. Raney Nickel 2800 (as purchased, a 50%by weight solution in water if used as purchased, Nickel >89% metalpresent) (6.8 g, 51.5 mmol) was added, and the flask sealed and flushedwith hydrogen. A 12″ needle was used to bubble hydrogen through thesolution for 10 minutes. The reaction was stirred for 5 days, using aballoon as a hydrogen reservoir. Hydrogen was also bubbled through thereaction mixture at 24 h and 48 h, 5 minutes each time. The metalcatalyst was then removed by filtering through Celite. The ethanolicsolution was concentrated, and 200 mL of DCM added to the resulting oil.The solution was washed with water (2×100 mL), dried over MgSO₄, andconcentrated. TLC indicated formation of the phytanol product, yield20.0 g.

Synthesis of Phytanyl Mesylate:

Phytanol (20.0 g, 66.7 mmol), triethylamine (18.6 mL, 133 mmol) and astir bar were added to a 1000 mL round bottom flask. The flask wassealed and flushed with nitrogen. Anhydrous DCM (250 mL) was added, andthe mixture cooled to −15° C. (Ice and NaCl). Mesyl Chloride (10.4 mL,133 mmol) was added slowly via syringe over a 30 minute period, and thereaction stirred at −15° C. for a further 1.5 hours. At this point TLCshowed that the starting material had been used up. The solution wasdiluted with DCM (250 mL) and washed with saturated NaHCO₃ (2×200 mL).The organic phase was then dried (MgSO₄), filtered and concentrated(rotovap). The crude product was purified by column chromatography.Yield 21.5 g, 85.7%.

Synthesis of Phytanyl Bromide:

Magnesium bromide etherate (17 g, 55 mmol) and a stir bar were added toa 500 mL round bottom flask. The flask was sealed and flushed withnitrogen and anhydrous diethyl ether (200 mL) added via cannula. Asolution of phytanyl mesylate (10.9 g, 28.9 mmol (FW=377)) in anhydrousether (50 mL) was also added via canulla, and the suspension stirredovernight. The following morning a precipitate had formed on the side ofthe flask. Chilled water (200 mL) was added (ppte dissolved) and themixture transferred to a 1000-mL separating funnel. After shaking, theorganic phase was separated. The aqueous phase was then extracted withether (2×150 mL) and all ether phases combined. The ether phase waswashed with water (2×150 mL), brine (150 mL) and dried over anhydrousMg₂SO₄. The solution was filtered, concentrated, and purified by flashchromatography. Final yield 9.5 g (26.3 mmol, 91.1%).

Synthesis of Compound A:

Magnesium turnings (720 mg, 30 mmol), a crystal of iodine, and a stirbarwere added to a 500 mL round-bottom flask. The flask was flushed withnitrogen and anhydrous diethyl ether (200 mL) added via cannula. Asolution of phytanyl bromide (9.5 g, 26.3 mmol) in anhydrous ether (20mL) was added and the resulting cloudy mixture refluxed overnight. Themixture was cooled to RT and, without removing the subaseal orcondenser, ethyl formate (2.2 g, 2.41 mL, 30 mmol) added via syringe and12″ needle. The addition was made dropwise, directly into the reactionmixture, and the cloudy suspension again stirred overnight. R.M. wastransferred to a 500-mL sep. funnel with ether (50 mL), and washed with10% H₂SO₄ (100 mL—the cloudy R.M. now clarified upon shaking), water(2×100 mL) and brine. The organic was dried over anhydrous Mg₂SO₄,filtered, and concentrated. Yield (crude) was 8 g. TLC indicated thatthe majority of product was the diphytanylmethyl formate, which waspurified by chromatography (0-6% ether in hexane).

Synthesis of Compound B:

The purified formate (A) (5.5 g, 8.86 mmol) was then transferred to a1000 mL round bottom flask with stirbar and 90% EtOH (500 mL) and KOH(2.0 g, 35.7 mmol) added. The reaction mixture was clear, and wasstirred overnight. The following day the mixture was concentrated byrotovap to 50% of its volume and then poured into 200 mL of 5% HCl. Theaqueous phase was extracted with ether (3×100 mL). The combined etherextracts were washed with water (3×200 mL), dried (MgSO₄), andconcentrated. TLC (DCM) revealed reaction to have gone cleanly tocompletion, and the product (5.5 g, 100%) was used without furtherpurification.

Synthesis of Compound C:

To a mixture of Compound B (5.5 g, 9.3 mmol), pyridinium chlorochromate(PCC) (5.5 g, 25.5 mmol) and anhydrous sodium carbonate (0.6 g, 5.66mmol) in DCM were added. The resulting suspension was stirred for 1 h,but TLC indicated still some starting material (SM) remaining. Thesuspension was stirred another hour, and appeared to have progressedslightly, but not to completion. Further PCC (1.0 g) and sodiumcarbonate (0.2 g) were added and the reaction stirred overnight.Reaction had now gone to completion. Ether (300 mL) was then added tothe mixture and the resulting brown suspension filtered through a pad ofsilica (300 mL), washing the pad with ether (3×100 mL). The ether phaseswere combined, concentrated, and purified to yield 5.0 g (90%) ofketone.

Synthesis of Compound D:

A 100 mL round bottom flask was charged with Compound C (1.4 g, 2.4mmol), 1,2,4-butanetriol (0.51 g, 4.8 mmol), pyridiniump-toluenesulfonate (0.06 g, 0.24 mmol), and a stir bar. The reactionvessel was flushed with nitrogen and anhydrous toluene (30 mL) added viacannula. The flask was equipped with a Dean-Stark tube and condenser andflushed with nitrogen. The reaction was refluxed under nitrogenovernight and progress of the reaction monitored via TLC. Afterrefluxing for three hours, reaction solution deposited in the Dean-Starktube was removed via syringe (20 mL) and the reaction vessel immediatelyreplenished with fresh toluene (20 mL). This was repeated every hour,for a total of three times, and then left to reflux mildly overnight.After cooling to room temperature, the reaction mixture was transferredto a 250 mL separatory funnel with toluene (2×5 mL), washed with 5%aqueous Na₂CO₃ (2×50 mL), water (50 mL), and dried over MgSO₄.Evaporation of the solvent gave 1.67 g of crude product which waspurified via column chromatography on silica gel (50 g) usingdichloromethane as eluent. Yield: 1.4 g, 2.06 mmol, 86%.

Synthesis of Compound E:

A 100 mL round bottom flask was charged with Compound D (1.4 g, 2.06mmol) and a stir bar. The vessel was flushed with nitrogen and DCM (25mL) added. Subsequently, triethylamine (0.72 g, 7.1 mmol, 0.99 mL) wasadded via syringe and the resulting solution cooled to −15° C. (NaCl,ice). In a separate 50 mL round bottom flask, a solution ofmethanesulfonic anhydride (0.74 g, 4.1 mmol) and DCM (20 mL) wasprepared. This solution was added drop wise to the above solution over a30 minute period. The reaction vessel was maintained at −15° C. Thereaction mixture was stirred at room temperature overnight and monitoredvia TLC. The reaction mixture was then diluted with DCM (25 mL), andwashed with NaHCO₃ (2×30 mL), then dried over anhydrous MgSO₄. The crudeproduct (1.7 g) was used in the next step without further purification.

Synthesis of DPan-C2K-DMA:

A 500 mL round bottom flask was charged with crude Compound E (1.7 g,2.5 mmol) and a stir bar. The reaction vessel was flushed with nitrogenand dimethylamine in THF (2.0 M, 65 mL) subsequently added via syringe.The resulting mixture was stirred for three days at room temperature.The reaction was concentrated and the crude product purified by columnchromatography using silica gel (40 g) with a gradient of 0-5% methanolin dichloromethane.

Synthesis of Compound F:

A 100 mL round bottom flask was charged with Compound C (1.2 g, 2.1mmol), 2-hydroxymethyl-1,3-propanediol (0.45 g, 4.2 mmol), pyridiniump-toluenesulfonate (0.05 g, 0.21 mmol), and a stir bar. The reactionvessel was flushed with nitrogen and anhydrous toluene (45 mL)subsequently added via cannula. The flask was equipped with a Dean-Starktube and condenser and flushed with nitrogen. The reaction was refluxedunder nitrogen overnight and progress of the reaction monitored via TLC.After refluxing for three hours, reaction solution deposited in theDean-Stark tube was removed via syringe (20 mL) and the reaction vesselimmediately replenished with fresh toluene (20 mL). This was repeatedevery hour, for a total of three times, and then left to reflux mildlyovernight. After cooling to room temperature, the reaction mixture wastransferred to a 250 mL separatory funnel with toluene (2×5 mL), washedwith 5% aqueous Na₂CO₃ (2×50 mL), water (50 mL), and dried over MgSO₄.Evaporation of the solvent gave 1.44 g of crude product which was thenpurified via column chromatography on silica gel (35 g) with 0-3%methanol gradient in dichloromethane.

Synthesis of Compound G:

A 250 mL round bottom flask was charged with Compound F (1.2 g, 1.8mmol) and a stir bar. The vessel was flushed with nitrogen and DCM (25mL) added. Subsequently, triethylamine (0.62 g, 6.1 mmol, 0.85 mL) wasadded via syringe and the resulting solution cooled to −15° C. (NaCl,ice). In a separate 50 mL round bottom flask, a solution ofmethanesulfonic anhydride (0.67 g, 3.7 mmol) and DCM (20 mL) wasprepared. This solution was added drop wise to the above solution over a30 minute period. The reaction vessel was maintained at −15° C. duringthe addition. The reaction mixture was stirred at room temperatureovernight and monitored via TLC. The reaction mixture was then dilutedwith DCM (25 mL) and washed with NaHCO₃ (2×30 mL), then dried overanhydrous MgSO₄. The crude product (1.6 g) was used in the followingstep without further purification.

Synthesis of DPan-C1K6-DMA:

A 250 mL round bottom flask was charged with crude Compound G (1.6 g,2.1 mmol) and a stir bar. The reaction vessel was flushed with nitrogenand dimethylamine in THF (2.0 M, 60 mL) subsequently added via syringe.The resulting mixture was stirred for six days at room temperature.After solvent was evaporated, the crude product was purified usingcolumn chromatography on silica gel (30 g) with 0-30% ethyl acetategradient in hexanes.

Synthesis of Compound H:

A 50 mL round bottom flask was charged with(R)-γ-hydroxymethyl-γ-butyrolactone (1.0 g, 8.6 mmol), flushed withnitrogen, and sealed with a rubber septum. Anhydrous THF (40 mL) wassubsequently added via syringe. The (R)-γ-hydroxymethyl-γ-butyrolactonesolution was then added drop wise under nitrogen to a prepared solutioncontaining LiAlH₄ (3.5 g, 92 mmol) in 160 mL anhydrous THF. During theaddition, the reaction vessel was maintained at 0° C. The resultingsuspension was stirred at room temperature overnight. The reactionmixture was cooled to 0° C. and brine (10-22 mL) added very slowly usinga Pasteur pipette. The mixture was stirred under nitrogen at roomtemperature overnight. The white solid was filtered and washed with THF(3×25 mL). The organics were combined and concentrated. After solventwas removed, the crude product seemed to contain water along with anoily residue; therefore, the crude product was azeotroped within ethanol(100 mL) resulting in a yellow oil. The crude product (0.45 g) was usedin the next step without further purification.

Synthesis of Compound I:

A 100 mL round bottom flask was charged with Compound C (1.0 g, 1.8mmol), Compound H (crude, 0.450 g, 3.6 mmol), pyridiniump-toluenesulfonate (0.05 g, 0.24 mmol), and a stir bar. The reactionvessel was flushed with nitrogen and anhydrous toluene (45 mL)subsequently added via cannula. The flask was equipped with a Dean-Starktube and condenser and flushed with nitrogen. The reaction was refluxedunder nitrogen overnight and progress of reaction monitored via TLC.After refluxing for three hours, reaction solution deposited in theDean-Stark tube was removed via syringe (20 mL) and the reaction vesselimmediately replenished with fresh toluene (20 mL). This was repeatedevery hour, for a total of five times, and then left to reflux mildlyovernight. After cooling to room temperature, the reaction mixture wastransferred to a 250 mL separatory funnel with toluene (2×5 mL), washedwith 5% aqueous Na₂CO₃ (2×50 mL), water (50 mL), and dried over MgSO₄.Evaporation of the solvent gave 1.13 g of crude product which was thenpurified via column chromatography on silica gel (30 g) usingdichloromethane as eluent. Yield, 1.0 g.

Synthesis of Compound J:

A 250 mL round bottom flask was charged with Compound I (1.0 g, 1.44mmol) and a stir bar. The vessel was flushed with nitrogen and DCM (25mL) added. Subsequently, triethylamine (0.51 g, 5 mmol, and 0.7 mL) wasadded via syringe and the resulting solution cooled to −15° C. (NaCl,ice). In a separate 50 mL round bottom flask, a solution ofmethanesulfonic anhydride (0.54 g, 3.0 mmol) and anhydrous DCM (20 mL)was prepared. This solution was added drop wise to the above solutionover a 30 minute period. The reaction vessel was maintained at −15° C.The reaction mixture was stirred at room temperature overnight andmonitored via TLC. The reaction mixture was then diluted with DCM (25mL) and washed with NaHCO₃ (2×30 mL), then dried over anhydrous MgSO₄.The crude product (1.2 g) was used in the next step without furtherpurification.

Synthesis of DPan-C3K-DMA:

A 100 mL round bottom flask was charged with crude Compound J (1.2 g,1.6 mmol) and a stir bar. The reaction vessel was flushed with nitrogenand dimethylamine in THF (2.0 M, 45 mL) subsequently added via syringe.The resulting mixture was stirred for four days at room temperature.After solvent was evaporated, the crude product was purified usingcolumn chromatography on silica gel (30 g) with 0-30% ethyl acetategradient in hexanes.

Example 8 Synthesis of DLen-C2K-DMA

DLen-C2K-DMA having the structure shown below was synthesized as shownin the following schematic diagram.

Synthesis of dilinolenyl ketone:

To a 1000 mL RBF containing a solution of dilinolenyl methanol (6.0 g,11.4 mmol) in anh. DCM (200 mL) was added pyridinium chlorochromate(7.39 g, 34.2 mmol), anh. sodium carbonate (1.0 g, 5.66 mmol) and astirbar. The resulting suspension was stirred under nitrogen at RT for 3h, after which time TLC indicated all SM to have been consumed. Ether(300 mL) was then added to the mixture and the resulting brownsuspension filtered through a pad of silica (300 mL), washing the padwith ether (3×100 mL). The ether phases were combined, concentrated andpurified to yield 4.2 g (8.0 mmol, 70%) of the ketone.

Synthesis of linolenyl ketal:

A 100 mL RBF was charged with dilinolenyl ketone (4.2 g, 8.2 mmol),1,2,4-butanetriol (3.4 g, 32 mmol), PPTS (200 mg, 0.8 mmol) and a stirbar. The flask was flushed with nitrogen and anhydrous toluene (60 mL)added. The reaction vessel was fitted with a Dean Stark tube andcondenser and brought to reflux and the reaction was left overnight.After cooling to room temperature, the reaction mixture diluted withtoluene (50 mL), and washed with 5% aq. Na₂CO₃ (2×50 mL), water (50 mL),dried (MgSO₄) and purified by chromatography to yield 3.0 g (4.9 mmol,59%) of the ketal.

Mesylate Formation:

A 250 mL RBF was charged with the linolenyl ketal (3.0 g, 4.9 mmol), TEA(2.2 mL, 15.6 mmol) and a stir bar. The flask was flushed with nitrogen,anh. DCM (20 mL) added, and the solution cooled to −15° C. In a separate50 mL flask, a solution of MsCl (9.7 mmol, 2 eqv.) in anhydrous DCM (30mL) was prepared, then transferred to the reaction vessel by syringeover 20 minutes. The reaction was stirred for 90 minutes at −15° C., atwhich point starting material had been consumed. The reaction mixturewas diluted with a further 50 mL of DCM, washed with NaHCO₃ (2×50 mL),dried (MgSO₄) and purified by chromatography. Final yield 3.1 g, 4.5mmol, 92%.

Synthesis of DLen-C2K-DMA:

A 250 mL RBF was charged with the mesylate (3.0 g, 4.35 mmol),isopropanol (25 mL) and a stir bar. The flask was flushed with nitrogen,sealed, and a 2.0 M solution of dimethylamine in methanol (120 mL) addedvia canulla. The reaction was stirred at room temperature for 3 days.The solution was concentrated and purified by chromatography. Finalyield 2.49 g, 3.9 mmol, 90%.

Example 9 Synthesis of γ-DLen-C2K-DMA

γ-DLen-C2K-DMA having the structure shown below was synthesized as shownin the following schematic diagram.

Synthesis of di-γ-linolenyl ketone:

To a 1000 mL RBF containing a solution of di-γ-linolenyl methanol (6.0g, 11.4 mmol) in anh. DCM (200 mL) was added pyridinium chlorochromate(7.39 g, 34.2 mmol), anh. sodium carbonate (1.0 g, 5.66 mmol) and astirbar. The resulting suspension was stirred under nitrogen at RT for 3h, after which time TLC indicated all SM to have been consumed. Ether(300 mL) was then added to the mixture and the resulting brownsuspension filtered through a pad of silica (300 mL), washing the padwith ether (3×100 mL). The ether phases were combined, concentrated andpurified to yield 5.5 g (10.5 mmol, 92%) of ketone.

Synthesis of γ-linolenyl ketal:

A 100 mL RBF was charged with di-γ-linolenyl ketone (2.14 g, 4.1 mmol),1,2,4-butanetriol (1.7 g, 16.0 mmol), PPTS (100 mg, 0.4 mmol) and a stirbar. The flask was flushed with nitrogen and anhydrous toluene (30 mL)added. The reaction vessel was fitted with a Dean Stark tube andcondenser and brought to reflux and the reaction was left overnight.After cooling to room temperature, the reaction mixture was washed with5% aq. Na₂CO₃ (2×50 mL), water (50 mL), dried (MgSO₄) and purified bychromatography to yield 1.34 g (2.2 mmol, 53%) of the ketal.

Mesylate Formation:

A 250 mL RBF was charged with the γ-linolenyl ketal (1.34 g, 2.19 mmol),TEA (1 mL, 7.1 mmol) and a stir bar. The flask was flushed withnitrogen, anh. DCM (10 mL) added, and the solution cooled to −15° C. Ina separate 50 mL flask, a solution of MsCl (342 μL, 4.4 mmol, 2 eqv.) inanhydrous DCM (15 mL) was prepared, then transferred to the reactionvessel by syringe over 20 minutes. The reaction was stirred for 90minutes at −15° C., at which point starting material had been consumed.The reaction mixture was diluted with a further 50 mL of DCM, washedwith NaHCO₃ (2×50 mL), dried (MgSO₄) and purified by chromatography.Final yield 1.31 g, 1.90 mmol, 87%.

Synthesis of γ-DLen-C2K-DMA:

A 250 mL RBF was charged with the mesylate (1.31 g, 1.9 mmol),isopropanol (10 mL) and a stir bar. The flask was flushed with nitrogen,sealed, and a 2.0 M solution of dimethylamine in methanol (60 mL) addedvia canulla. The reaction was stirred at room temperature for 3 days.The solution was concentrated and purified by chromatography. Finalyield 1.1 g, 1.72 mmol, 91%.

Example 10 Lipid Encapsulation of siRNA

All siRNA molecules used in these studies were chemically synthesizedand annealed using standard procedures.

In some embodiments, siRNA molecules were encapsulated into serum-stablenucleic acid-lipid particles (SNALP) composed of the following lipids:(1) the lipid conjugate PEG2000-C-DMA (3-N-[(-methoxypoly(ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine); (2) one or morecationic lipids or salts thereof (e.g., cationic lipids of Formula I-XIVand/or other cationic lipids described herein); (3) the phospholipidDPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) (Avanti Polar Lipids;Alabaster, Ala.); and (4) synthetic cholesterol (Sigma-Aldrich Corp.;St. Louis, Mo.) in the molar ratio 1.4:57.1:7.1:34.3, respectively. Inother words, siRNA molecules were encapsulated into SNALP of thefollowing “1:57” formulation: 1.4% PEG2000-C-DMA; 57.1% cationic lipid;7.1% DPPC; and 34.3% cholesterol. It should be understood that the 1:57formulation is a target formulation, and that the amount of lipid (bothcationic and non-cationic) present and the amount of lipid conjugatepresent in the formulation may vary. Typically, in the 1:57 formulation,the amount of cationic lipid will be 57.1 mol % f 5 mol %, and theamount of lipid conjugate will be 1.4 mol % f 0.5 mol %, with thebalance of the 1:57 formulation being made up of non-cationic lipid(e.g., phospholipid, cholesterol, or a mixture of the two).

For vehicle controls, empty particles with identical lipid compositionmay be formed in the absence of siRNA.

Example 11 Characterization of Novel ApoB SNALP Formulations ContainingVarious Cationic Lipids

This example demonstrates the efficacy of novel SNALP formulationscontaining cationic lipids described herein with an siRNA targeting APOBin a mouse liver model. The APOB siRNA sequence used in this study isprovided in Table 1.

TABLE 1 % 2′OMe- % Modified siRNA APOB siRNA Sequence Modifiedin DS Region ApoB-10164 5′-AGU G UCA U CACAC U GAAUACC-3′ (SEQ ID NO: 1)7/42 = 16.7% 7/38 = 18.4% 3′-GU U CACAGUAGU G U G AC U UAU-5′(SEQ ID NO: 2) Column 1: The number after “ApoB” refers to thenucleotide position of the 5′ base of the sense strand relative to thehuman APOB mRNA sequence NM_000384. Column 2: 2′OMe nucleotides areindicated in bold and underlined. The 3′-overhangs on one or bothstrands of the siRNA molecule may alternatively comprise 1-4deoxythymidine (dT) nucleotides, 1-4 modified and/or unmodified uridine(U) ribonucleotides, or 1-2 additional ribonucleotides havingcomplementarity to the target sequence or the complementary strandthereof. Column 3: The number and percentage of 2′OMe-modifiednucleotides in the siRNA molecule are provided. Column 4: The number andpercentage of modified nucleotides in the double-stranded (DS) region ofthe siRNA molecule are provided.

1:57 SNALP formulations containing encapsulated APOB siRNA were preparedas described in Section V above with the following cationic lipids: (1)DLinDMA; (2) DLin-K-C2-DMA (“C2K”); (3) DLin-K-C3-DMA (“C3K”); (4)DLin-K-C4-DMA (“C4K”); (5) DLin-K6-DMA; (6) DLin-C2-DMA; (7) DLenDMA;(8) γ-DLenDMA (“g-DLenDMA”); (9) DLin-K-DMA; (10) DLinMorph; (11)Linoleyl/Oleyl DMA (“Lin/Ol”); (12) Linoleyl/Linolenyl DMA (“Lin/Len”);(13) Linoleyl/Phytanyl DMA (“Lin/Pan”); (14) Linoleyl/Stearyl DMA(“Lin/Str”); and (15) Linoleyl/C6:1 DMA (“Lin/C6:1”).

Each SNALP formulation was administered by intravenous (IV) injection at0.1 mg/kg into female BALB/c mice (n=3 per group). Plasma totalcholesterol and/or liver ApoB mRNA levels were evaluated at 48 hoursafter SNALP administration. For dose response studies, SNALPformulations were administered by IV injection at 0.01 mg/kg, 0.03mg/kg, or 0.1 mg/kg into female Balb/c mice (n=3 per group). Liver ApoBmRNA levels were evaluated at 48 hours after SNALP administration.

FIGS. 1-3 show a comparison of the plasma total cholesterol knockdownefficacy and/or the liver ApoB mRNA knockdown activity of each of theseSNALP formulations (Error bars=SD). FIG. 4 shows a dose responseevaluation of three different doses of SNALP formulations containingeither DLinDMA, DLin-K-C2-DMA (“C2K”), DLenDMA, or γ-DLenDMA on liverApoB mRNA knockdown activity (Error bars=SD).

These figures illustrate that SNALP formulations containing eitherDLin-K-C2-DMA (“C2K”) or γ-DLenDMA were unexpectedly more potent insilencing ApoB expression in vivo compared to SNALP formulationscontaining either DLinDMA or DLenDMA. These figures also illustrate thata SNALP formulation containing an asymmetric cationic lipid such asLinoleyl/Linolenyl DMA (“Lin/Len”) displayed greater ApoB silencingactivity compared to a SNALP formulation containing DLinDMA.

Example 12 Characterization of Additional Novel ApoB SNALP FormulationsContaining Various Cationic Lipids

This example demonstrates the efficacy of additional novel SNALPformulations containing cationic lipids described herein with an siRNAtargeting APOB in a mouse liver model. The APOB siRNA sequence used inthese studies is provided in Table 1.

1:57 SNALP formulations containing encapsulated APOB siRNA at a 6:1lipid:drug (L:D) ratio were prepared with the following cationic lipids:(1) DLinDMA; (2) Linoleyl/Linolenyl DMA (“Lin/LenDMA”); (3) DPanDMA; (4)TLinDMA; (5) Linoleyl/C6:0 DMA (“Lin/6:0”); (6) C2-DPanDMA; (7)DLin-C2K-Pip (3OH); and (8) DHep-C2K-DMA.

Each SNALP formulation was administered by intravenous (IV) injection at0.1 mg/kg into female Balb/c mice (n=3 per group). Livers were collectedat 48 hours after SNALP administration and liver ApoB mRNA levels wereevaluated by performing an ApoB/GAPDH QG assay. Table 2 provides acharacterization of the SNALP formulations used in this in vivo study.

TABLE 2 SNALP Size (nm) Poly Encapsulation % 1:57 DLinDMA 74.96 0.023 7977.88 0.054 1:57 Lin/LenDMA 81.12 0.023 63 98.21 0.010 1:57 DPanDMA64.84 0.063 78 65.44 0.033 1:57 TLinDMA 71.98 0.037 51 73.12 0.069 1:57Lin/6:0 101.7 0.095 78 97.64 0.119 1:57 C2-DPanDMA 95.09 0.058 90 98.260.052 1:57 DLin-C2K-Pip (3OH) 68.92 0.093 73 73.27 0.067 1:57DHep-C2K-DMA 75.29 0.034 89 79.89 0.037 1:57 DLinDMA (TFU) 82.23 0.03989 Columns 2 & 3: The bottom value in each entry corresponds to theparticle size and polydispersity observed 10 days after the formulationwas prepared.

FIG. 5 illustrates, inter alia, that: (1) DHep-C2K-DMA, which containsdouble bonds in the trans configuration thought to reduce potency, wasunexpectedly comparable to DLinDMA with respect to silencing activity;(2) C2-DPanDMA, which contains saturated fatty alkyl chains thought todecrease potency, was unexpectedly more potent compared to DLinDMA andsubstantially more potent compared to DPanDMA with respect to silencingactivity; (3) TLinDMA displayed silencing activity that was comparableto DLinDMA; and (4) Lin/LenDMA had more potent silencing activity thanDLinDMA. A similar study with 1:57 SNALP containing C2-TLinDMA showedthat C2-TLinDMA (49% knockdown) was more potent than DLinDMA (25%knockdown) with respect to ApoB silencing activity.

In another study, 1:57 SNALP formulations containing encapsulated APOBsiRNA at a 6:1 L:D ratio were prepared with the following cationiclipids: (1) DLinDMA; (2) C2-DPanDMA; (3) DPan-C2K-DMA; (4) DPan-C3K-DMA;and (5) DPan-C1K6-DMA.

Each SNALP formulation was administered by intravenous (IV) injection at0.1 mg/kg into female Balb/c mice (n=3 per group). Livers were collectedat 48 hours after SNALP administration and liver ApoB mRNA levels wereevaluated by performing an ApoB/GAPDH QG assay. Table 3 provides acharacterization of the SNALP formulations used in this in vivo study.

TABLE 3 Size (nm) Poly Encapsulation % 1:57 DLinDMA 76.63 0.033 81 1:57C2-DPanDMA 85.80 0.003 88 1:57 DPan-C2K-DMA 79.06 0.020 87 1:57DPan-C3K-DMA 93.46 0.002 90 1:57 DPan-C1K6-DMA 72.78 0.031 77

FIG. 6 illustrates that C2-DPanDMA, DPan-C2K-DMA, and DPan-C3K-DMA,which contains saturated fatty alkyl chains thought to decrease potency,were unexpectedly more potent compared to DLinDMA with respect tosilencing activity. C2-DPanDMA had the greatest activity of all thephytanyl-containing cationic lipids tested.

Example 13 Characterization of Additional Novel ApoB SNALP FormulationsContaining Various Cationic Lipids

This example demonstrates the efficacy of additional novel SNALPformulations containing cationic lipids described herein with an siRNAtargeting APOB in a mouse liver model. The APOB siRNA sequence used inthese studies is provided in Table 1.

1:57 SNALP formulations containing encapsulated APOB siRNA were preparedas described in Section V above with the following cationic lipids: (1)DLin-C2K-DMA (“C2K”); (2) γ-DLen-C2K-DMA (“g-DLen-C2K-DMA”); and (3)DLen-C2K-DMA.

For dose response studies, SNALP formulations were administered by IVinjection at 0.01 mg/kg, 0.033 mg/kg, or 0.1 mg/kg into female Balb/cmice (n=3 per group). Liver ApoB mRNA levels were evaluated at 48 hoursafter SNALP administration by a branched DNA assay (QuantiGene assay) toassess ApoB mRNA relative to the housekeeping gene GAPDH.

FIG. 7 shows a comparison of the liver ApoB mRNA knockdown activity ofeach of these SNALP formulations at three different doses (Errorbars=SD), as well as the KD50 values obtained for each of theseformulations. In particular, FIG. 7 shows that a SNALP formulationcontaining g-DLen-C2K-DMA displayed similar ApoB silencing activity atall three doses and an identical KD50 value compared to a SNALPformulation containing C2K. Furthermore, FIG. 7 shows that a SNALPformulation containing DLen-C2K-DMA displayed considerable potency insilencing ApoB mRNA expression.

Example 14 Increased Potency of Novel C2K SNALP Formulation at SilencingApoB Expression

This example further demonstrates the surprising increase in potencyobserved for the DLin-K-C2-DMA (“C2K”) SNALP formulation at silencingApoB expression in both mouse and rat liver models. The APOB siRNAsequence used in these studies is provided in Table 1 above.

1:57 SNALP formulations containing encapsulated APOB siRNA were preparedas described in Section V above with either C2K or DLinDMA as thecationic lipid component. APOB C2K SNALP formulations were administeredby intravenous (IV) injection at 0.01 mg/kg, 0.05 mg/kg, or 0.25 mg/kginto female Balb/c mice (n=4 per group), while APOB DLinDMA SNALPformulations were administered by IV injection at 0.05 mg/kg, 0.10mg/kg, or 0.25 mg/kg into female Balb/c mice (n=4 per group). For therat study, C2K or DLinDMA SNALP formulations were administered by IVinjection at 0.25 mg/kg into Sprague Dawley rats. Liver ApoB mRNA levelswere evaluated at 48 hours after SNALP administration.

FIG. 8 shows a dose response evaluation of three different doses ofSNALP formulations containing either DLinDMA or C2K on liver ApoB mRNAknockdown activity in mice (Error bars=SD). FIG. 9 shows thereproducibility of the dose response study in mice using two independentSNALP batches. FIG. 10 shows that the improved liver ApoB mRNA knockdownactivity of C2K SNALP versus DLinDMA SNALP is preserved in rats.

Importantly, these figures illustrate that the C2K SNALP formulation wasabout 5 times more potent than a corresponding DLinDMA SNALP formulationbased on the KD₅₀ for liver ApoB mRNA silencing in mice. These figuresalso illustrate that the degree of ApoB mRNA silencing at 0.25 mg/kg forboth formulations is comparable between rat and mouse.

Example 15 Characterization of Inflammatory Response to APOB DLinDMA andC2K SNALP Formulations in Human Whole Blood

Inflammatory response to DLinDMA or C2K SNALPs containing APOB siRNA wasevaluated by measuring cytokine induction ex vivo in whole blood samplestaken from human subjects. For both the DLinDMA and C2K formulations,the SNALPs contained either no siRNA payload (“empty”) or APOB siRNApayload. The APOB siRNAs tested included “ApoB-8” (the APOB siRNAexemplified in Examples 12-15 and Table 1), “2/5” (described in Table4), “3/5” (described in Table 4), and “6/5” (described in Table 4).Briefly, fresh blood was isolated, immediately diluted 1:1 with 0.9%saline solution, and plated 0.45 mL/well into 48 well tissue culturetreated plates. SNALPs were diluted in formulation PBS and added to theplated blood samples at a concentration of either 300 nM or 1200 nM.After 24 hours, the plates were centrifuged at 1200 rpm for 20 minutesand the supernatant (plasma) collected. Cytokine induction was measuredby ELISA and/or Cytometric Bead Array.

FIGS. 11 and 12 show the results of cytokine induction assays for threedonors at increasing SNALP concentrations. FIG. 11 shows thatinflammatory response to APOB SNALP formulations, as measured by theconcentration of the cytokine TNF, was significantly higher for SNALPDLinDMA formulations than for SNALP C2K formulations for two of thethree donors. Additionally, all three donors exhibited significantlyless inflammatory response to the APOB siRNAs 2/5, 3/5, and 6/5 ascompared to the APOB siRNA ApoB-8. Similarly, FIG. 12 shows that theDLinDMA formulation induces a stronger IL-8 cytokine response than theC2K formulation, as measured by ELISA. Moreover, the APOB siRNAs 3/5 and6/5 in C2K SNALPs generally induced less of an immunostimulatoryresponse than did the ApoB-8 siRNA in C2K SNALPs. These figuresdemonstrate that the C2K SNALP formulation is less immunostimulatorythan the DLinDMA SNALP formulation. Additionally, these figuresdemonstrate that increasing the number of selective 2′OMe modificationsto the siRNA sequence (e.g., 2′OMe modifications at G's and/or U's inthe double-stranded and/or 3′ overhang regions of the siRNA sequence)can decrease the immunostimulatory response to the siRNA.

TABLE 4 % 2′OMe- % Modified siRNA APOB siRNA Sequence Modifiedin DS Region 2/5 5′-AG U G U CA U CACAC U GAA U ACC-3′ (SEQ ID NO: 3)12/42 = 28.6% 10/38 = 26.3% 3′- GUU CACAG U AG U G U GAC U UAU-5′(SEQ ID NO: 11) 3/5 5′-AG U G U CA U CACAC UG AA U ACC-3′ (SEQ ID NO: 4)13/42 = 31.0% 11/38 = 28.9% 3′- GUU CACAG U AG U G U GAC U UAU-5′(SEQ ID NO: 11) 6/5 5′-A GU G U CA U CACAC UG AA U ACC-3′ (SEQ ID NO: 7)14/42 = 33.3% 12/38 = 31.6% 3′- GUU CACAG U AG U G U GAC U UAU-5′(SEQ ID NO: 11) Column 1: “2/5,” “3/5,” and “6/5” refer to APOB sensestrand annealed to antisense strand (e.g., sense strand 2 annealed toantisense strand 5 = 2/5). Column 2: 2′OMe nucleotides are indicated inbold and underlined. The 3'-overhangs on one or both strands of thesiRNA molecule may alternatively comprise 1-4 deoxythymidine (dT)nucleotides, 1-4 modified and/or unmodified uridine (U) ribonucleotides,or 1-2 additional ribonucleotides having complementarity to the targetsequence or the complementary strand thereof. Column 3: The number andpercentage of 2′OMe-modified nucleotides in the siRNA molecule areprovided. Column 4: The number and percentage of modified nucleotides inthe double-stranded (DS) region of the siRNA molecule are provided.

Example 16 In Vitro and In Vivo Activity Screen of Modified APOB siRNAsin C2K SNALPs

As shown in FIGS. 11 and 12, APOB siRNAs which have the same nucleotidesequence as ApoB-8 but which have an increased number of modifiednucleotides are less immunostimulatory than ApoB-8. This exampledemonstrates that APOB siRNAs which have the same nucleotide sequence asApoB-8 but which have an increased number of modified nucleotides are atleast as effective as ApoB-8 in knocking down ApoB mRNA expression.

APOB siRNAs of the same nucleotide sequence as ApoB-8 (exemplified inExamples 12-15 and Table 1, and also called “ApoB-10164”) were modifiedto incorporate an increasing number and alternate patterns of 2′OMenucleotides. Six different sense strands (S-1 to S-6) and six differentantisense strands (AS-1 to AS-6) were designed. Sense strand 1 (S-1) isthe same pattern of modification as the ApoB-8 sense strand (SEQ IDNO:1), and antisense strand 1 (AS-1) is the same pattern of modificationas the ApoB-8 antisense strand (SEQ ID NO:2), and were generated assynthesis controls. APOB double-stranded siRNAs were generated by mixand match annealing of sense strands 2-6 (S-2 to S-6) and antisensestrands 2-6 (AS-2 to AS-6). Compared to siApoB-8 (also referred to inthis example as “1/1”), the number of modifications for double-strandedAPOB siRNAs increased from 7 to about 9-12 in the double-strandedregion. Additionally, some of the patterns of modification include2′OMe-modified nucleotides in the 3′ overhang of one or both strands ofthe siRNA, such that the number of modifications are further increasedto about 10-14 in the entire siRNA molecule. Table 5 shows modified APOBsense strands 1-6 (S-1 to S-6), modified ApoB antisense strands 1-6(AS-1 to AS-6), and the double-stranded APOB siRNAs that resulted fromthe mix and match annealing of S-2 to S-6 with AS-2 to AS-6.

TABLE 5 % 2′-OMe- % Modified siRNA APOB siRNA Sequence Modifiedin DS Region S-1 5′-AGU G UCA U CACAC U GAAUACC-3′ (SEQ ID NO: 1) 3/21 = 14.3%  3/19 = 15.8% S-2 5′-AG U G U CA U CACAC U GAA U ACC-3′(SEQ ID NO: 3)  5/21 = 23.8%  5/19 = 26.3% S-3 5′-AG U G U CA U CACAC UGAA U ACC-3′ (SEQ ID NO: 4)  6/21 = 28.6%  6/19 = 31.6% S-4 5′-A G U GUCA U CACACU G AA U ACC-3′ (SEQ ID NO: 5)  5/21 = 23.8%  5/19 = 26.3%S-5 5′-A GUGU CA U CACAC U GAA U ACC-3′ (SEQ ID NO: 6)  7/21 = 33.3% 7/19 = 36.8% S-6 5′-A GU G U CA U CACAC UG AA U ACC-3′ (SEQ ID NO: 7) 7/21 = 33.3%  7/19 = 36.8% AS-1 5′-UAU U CA G U G UGAUGACAC U UG-3′(SEQ ID NO: 2)  4/21 = 19.0%  4/19 = 21.1% AS-2 5′-UAU U CAG U G U GA UGACAC U UG-3′ (SEQ ID NO: 8)  5/21 = 23.8%  5/19 = 26.3% AS-3 5′-UAU UCA G U G U G AUGACAC U UG-3′ (SEQ ID NO: 9)  5/21 = 23.8%  5/19 = 26.3%AS-4 5′-UAU U CA G U G UGAUGACAC UUG -3′ (SEQ ID NO: 10)  6/21 = 28.6% 4/19 = 21.1% AS-5 5′-UAU U CAG U G U GA U GACAC UUG -3′ (SEQ ID NO: 11) 7/21 = 33.3%  5/19 = 26.3% AS-6 5′-UAU U CA G U G U G AUGACAC UUG -3′(SEQ ID NO: 12)  7/21 = 33.3%  5/19 = 26.3% 1/1 5′-AGU G UCA U CACAC UGAAUACC-3′ (SEQ ID NO: 1)  7/42 = 16.7%  7/38 = 18.4% 3′-GU U CACAGUAGUG U G AC U UAU-5′ (SEQ ID NO: 2) 2/2 5′-AG U G U CA U CACAC U GAA UACC-3′ (SEQ ID NO: 3) 10/42 = 23.8% 10/38 =  26.3% 3′-GU U CACAG U AG UG U GAC U UAU-5′ (SEQ ID NO: 8) 2/3 5′-AG U G U CA U CACAC U GAA UACC-3′ (SEQ ID NO: 3) 10/42 = 23.8% 10/38 = 26/3% 3′-GU U CACAGUA G U GU G AC U UAU-5′ (SEQ ID NO: 9) 3/2 5′-AG U G U CA U CACAC UG AA U ACC-3′(SEQ ID NO: 4) 11/42 = 26.2% 11/38 = 28.9% 3′-GU U CACAG U AG U G U GACU UAU-5′ (SEQ ID NO: 8) 3/3 5′-AG U G U CA U CACAC UG AA U ACC-3′(SEQ ID NO: 4) 11/42 = 26.2% 11/38 = 28.9% 3′-GU U CACAGUA G U G U G ACU UAU-5′ (SEQ ID NO: 9) 4/2 5′-A G U G UCA U CACACU G AA U ACC-3′(SEQ ID NO: 5) 10/42 = 23.8% 10/38 = 26.3% 3′-GU U CACAG U AG U G U GACU UAU-5′ (SEQ ID NO: 8) 4/3 5′-A G U G UCA U CACACU G AA U ACC-3′(SEQ ID NO: 5) 10/42 = 23.8% 10/38 = 26.3% 3′-GU U CACAGUA G U G U G ACU UAU-5′ (SEQ ID NO: 9) 5/2 5′-A GUGU CA U CACAC U GAA U ACC-3′(SEQ ID NO: 6) 12/42 = 28.6 12/38 = 31.6% 3′-GU U CACAG U AG U G U GAC UUAU-5′ (SEQ ID NO: 8) 5/3 5′-A GUGU CA U CACAC U GAA U ACC-3′(SEQ ID NO: 6) 12/42 = 28.6 12/38 = 31.6% 3′-GU U CACAGUA G U G U G AC UUAU-5′ (SEQ ID NO: 9) 6/2 5′-A GU G U CA U CACAC UG AA U ACC-3′(SEQ ID NO: 7) 12/42 = 28.6 12/38 = 31.6% 3′-GU U CACAG U AG U G U GAC UUAU-5′ (SEQ ID NO: 8) 6/3 5′-A GU G U CA U CACAC UG AA U ACC-3′(SEQ ID NO: 7) 12/42 = 28.6 12/38 = 31.6% 3′-GU U CACAGUA G U G U G AC UUAU-5′ (SEQ ID NO: 9) 2/4 5′-AG U G U CA U CACAC U GAA U ACC-3′(SEQ ID NO: 3) 11/42 = 26.2%  9.38 = 23.7% 3′- GUU CACAGUAGU G U G AC UUAU-5′ (SEQ ID NO: 10) 2/5 5′-AG U G U CA U CACAC U GAA U ACC-3′(SEQ ID NO: 3) 12/42 = 28.6% 10/38 = 26.3% 3′- GUU CACAG U AG U G U GACU UAU-5′ (SEQ ID NO: 11) 2/6 5′-AG U G U CA U CACAC U GAA U ACC-3′(SEQ ID NO: 3) 12/42 = 28.6% 10/38 = 26.3% 3′- GUU CACAGUA G U G U G ACU UAU-5′ (SEQ ID NO: 12) 3/4 5′-AG U G U CA U CACAC UG AA U ACC-3′(SEQ ID NO: 4) 12/42 = 28.6% 10/38 = 26.3% 3′- GUU CACAGUAGU G U G AC UUAU-5′ (SEQ ID NO: 10) 3/5 5′-AG U G U CA U CACAC UG AA U ACC-3′(SEQ ID NO: 4) 13/42 = 31.0% 11/38 = 28.9% 3′- GUU CACAG U AG U GUGAC UUAU-5′ (SEQ ID NO: 11) 3/6 5′-AG U G U CA U CACAC UG AA U ACC-3′(SEQ ID NO: 4) 13/42 = 31.0% 11/38 = 28.9% 3′- GUU CACAGUA G U G U G ACU UAU-5′ (SEQ ID NO: 12) 4/4 5′-A G U G UCA U CACACU G AA U ACC-3′(SEQ ID NO: 5) 11/42 = 26.2%  9.38 = 23.7% 3′- GUU CACAGUAGU G U G AC UUAU-5′ (SEQ ID NO: 10) 4/5 5′-A G U G UCA U CACACU G AA U ACC-3′(SEQ ID NO: 5) 12/42 = 28.6% 10/38 = 26.3% 3′- GUU CACAG U AG U G U GACU UAU-5′ (SEQ ID NO: 11) 4/6 5′-A G U G UCA U CACACU G AA U ACC-3′(SEQ ID NO: 5) 12/42 = 28.6% 10/38 = 26.3% 3′- GUU CACAGUA G U G U G ACU UAU-5′ (SEQ ID NO: 12) 5/4 5′-A GUGU CA U CACAC U GAA U ACC-3′(SEQ ID NO: 6) 13/42 = 31.0% 11/38 = 28.9% 3′- GUU CACAGUAGU G U G AC UUAU-5′ (SEQ ID NO: 10) 5/5 5′-A GUGU CA U CACAC U GAA U ACC-3′(SEQ ID NO: 6) 14/42 = 33.3% 12/38 = 31.6% 3′- GUU CACAG U AG U G U GACU UAU-5′ (SEQ ID NO: 11) 5/6 5′-A GUGU CA U CACAC U GAA U ACC-3′(SEQ ID NO: 6) 14/42 = 33.3% 12/38 = 31.6% 3′- GUU CACAGUA G U G U G ACU UAU-5′ (SEQ ID NO: 12) 6/4 5′-A GU G U CA U CACAC UG AA U ACC-3′(SEQ ID NO: 7) 13/42 = 31.0% 11/38 = 28.9% 3′- GUU CACAGUAGU G U G AC UUAU-5′ (SEQ ID NO: 10) 6/5 5′-A GU G U CA U CACAC UG AA U ACC-3′(SEQ ID NO: 7) 14/42 = 33.3% 12/38 = 31.6% 3′- GUU CACAG U AG U G U GACU UAU-5′ (SEQ ID NO: 11) 6/6 5′-A GU G U CA U CACAC UG AA U ACC-3′(SEQ ID NO: 7) 14/42 = 33.3% 12/38 = 31.6% 3′- GUU CACAGUA G U G U G ACU UAU-5′ (SEQ ID NO: 12) Column 1: Sense strand, antisense strand, orsense strand/antisense strand. APOB sense strands 1-6 and antisensestrands 1-6 were designed with alternate patterns of modification. APOBsense strands 2-6 were mix and match annealed to APOB antisense strands2-6 (e.g., sense strand 2 annealed to antisense strand 5 = 2/5). 1/1,which is the same as ApoB-10164 in Example 5, was a synthesis control.Column 2: 2′OMe nucleotides are indicated in bold and underlined. The3′-overhangs on one or both strands of the siRNA molecule mayalternatively comprise 1-4 deoxythymidine (dT) nucleotides, 1-4 modifiedand/or unmodified uridine (U) ribonucleotides, or 1-2 additionalribonucleotides having complementarity to the target sequence or thecomplementary strand thereof. Column 3: The number and percentage of2′OMe-modified nucleotides in the siRNA molecule are provided. Column 4:The number and percentage of modified nucleotides in the double-stranded(DS) region of the siRNA molecule are provided.

1:57 SNALP formulations containing encapsulated APOB duplexes asdescribed in Table 5 were prepared at 3 mg scale with the cationic lipidDLin-C2K-DMA. For the in vitro assays, transfections of human primaryhepatocytes were performed on Primaria plates according to standardprotocols using a SNALP dose range of 0.125-0.00781 μg/mL. Cells wereplated at 50,000 cells/well and incubated overnight at 37° C. Attransfection, SNALP was diluted to the desired dose and pre-incubatedwith serum at 37° C. for 1 hour, then the cell media was replaced with80 μL fresh media and 20 μL pre-incubated SNALP. The cells wereincubated with SNALP for 24 hours, then the media was removed and thecells lysed for QuantiGene Analysis. Quantitation of mRNA levels wasaccomplished using individual standard curves for 1/1-C2K and 2/5-C2K.The remaining SNALPs were quantitated against the 1/1-C2K curve, whichresulted in differences of up to 12% in the actual dose that wasadministered; therefore, FIGS. 13 and 14 also depict the actual doseadministered for a SNALP where the dose varied from the 0.125 μg/mLintended dose.

FIG. 13 shows the knockdown efficiency in human primary hepatocytes fromC2K SNALPs comprising the modified APOB siRNA sequences of Table 5(n=2). Surprisingly, the APOB mRNA knockdown activity of exemplary2′OMe-modified APOB SNALP formulations containing C2K was similar to orgreater than the silencing activity observed with the 1/1-C2K SNALPformulation (i.e., the ApoB-8 SNALP). The results show that increasingthe number of modifications, from 7 in ApoB-8 to up to 14 in some of themodified APOB siRNAs, does not decrease activity, and in some casesincreases silencing activity.

FIG. 14 shows a comparison of in vitro silencing activity of selectedmodified APOB siRNAs for different SNALP formulations (DLinDMA vs.DLin-C2K-DMA as the cationic lipid). Silencing activity is measured asthe percentage of ApoB mRNA expression relative to transfection with PBScontrol. Overall, there was increased silencing activity with C2K SNALPsthan with DLinDMA SNALPs. In particular, 2/5-C2K and 3/4-C2K exhibitedgreater silencing activity than 1/1-C2K (i.e., the ApoB-8 SNALP).

Next, 1:57 SNALP formulations comprising the APOB siRNAs 1/1, 2/2, 2/5,3/2, 3/5, 4/2, 4/5, or 6/5 and DLin-C2K-DMA were utilized to assesssilencing activity in vivo in mice. Each SNALP formulation wasadministered by intravenous (IV) bolus injection at 0.01, 0.02, or 0.05mg/kg into female BALB/c mice (n=4 per group). Liver ApoB mRNA levelswere evaluated at 48 hours after SNALP administration by QuantiGeneAnalysis. Quantitation of mRNA levels was accomplished using individualstandard curves for 1/1-C2K and 2/5-C2K. The remaining SNALPs werequantitated against the 1/1-C2K curve, which resulted in differences ofup to 18% in the actual dose that was administered; therefore, FIGS. 15and 16 also depict the percentage difference in the actual doseadministered for a SNALP where the dose varied from the intended dose.

FIG. 15 shows the silencing activity of 0.02 mg/kg of 1:57 DLin-C2K-DMAformulated modified APOB SNALPs. All of the modified APOB SNALPs aregenerally as effective as 1/1-C2K (i.e., the ApoB-8 SNALP) in silencingApoB expression. In particular, 3/5-C2K showed the greatest silencingactivity of all the modified SNALPs tested.

FIG. 16 shows a comparison of in vivo silencing activity for selectedmodified APOB siRNAs in different SNALP formulations (DLinDMA vs.DLin-C2K-DMA as the cationic lipid). Surprisingly, although the modifiedAPOB siRNA sequences all generally were at least as effective as 1/1(i.e., ApoB-8) in both the DLinDMA and DLin-C2K-DMA formulations,different modified APOB siRNA sequences were most effective for thedifferent cationic lipid compositions. In particular, siRNA sequence 2/5showed the greatest silencing activity in DLinDMA, but siRNA sequence3/5 showed the greatest silencing activity in DLin-C2K-DMA.

FIG. 17 shows the silencing activity of APOB siRNA 1/1 (“siApoB-8”) orAPOB siRNA 3/5 (“siApoB-10”) formulated in 1:57 SNALP containing DLinDMAor DLin-C2K-DMA (“C2K”). For these dose response studies, SNALPformulations were administered by IV injection at 0.05, 0.10, or 0.25mg/kg for DLinDMA and at 0.01, 0.025, or 0.05 mg/kg for C2K into femaleBalb/c mice (n=4 per group). Liver ApoB mRNA levels were evaluated at 48hours after SNALP administration (Error bars=SD). In particular, FIG. 17shows that DLinDMA SNALP formulations containing either siApoB-8 orsiApoB-10 displayed similar silencing activities based on the KD₅₀ forliver ApoB mRNA silencing in mice. Similarly, FIG. 17 shows that C2KSNALP formulations containing either siApoB-8 or siApoB-10 displayedsimilar silencing activities based on the KD₅₀ for liver ApoB mRNAsilencing in mice. Notably, C2K SNALP formulations containing eithersiApoB-8 or siApoB-10 were significantly more potent than thecorresponding DLinDMA SNALP formulations based on a comparison of theirKD₅₀ values.

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reading the above description. The scopeof the invention should, therefore, be determined not with reference tothe above description, but should instead be determined with referenceto the appended claims, along with the full scope of equivalents towhich such claims are entitled. The disclosures of all articles andreferences, including patent applications, patents, PCT publications,and Genbank Accession Nos., are incorporated herein by reference for allpurposes.

1. A nucleic acid-lipid particle comprising: (a) an interfering RNA thatsilences Apolipoprotein B (APOB) expression; (b) a cationic lipid havingthe following structure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently optionally substituted C₁₂-C₂₄ alkyl, optionallysubstituted C₁₂-C₂₄ alkenyl, optionally substituted C₁₂-C₂₄ alkynyl, oroptionally substituted C₁₂-C₂₄ acyl; R³ and R⁴ are either the same ordifferent and are independently optionally substituted C₁-C₆ alkyl,optionally substituted C₂-C₆ alkenyl, or optionally substituted C₂-C₆alkynyl or R³ and R⁴ may join to form an optionally substitutedheterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosenfrom nitrogen and oxygen; R⁵ is either absent or hydrogen or C₁-C₆ alkylto provide a quaternary amine; m, n and p are either the same ordifferent and are independently either 0, 1 or 2, with the proviso thatm, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; Y and Z areeither the same or different and are independently O, S, or NH; and (c)a non-cationic lipid.
 2. The nucleic acid-lipid particle in accordancewith claim 1, wherein at least one of R¹ and R² has at least two sitesof unsaturation.
 3. (canceled)
 4. The nucleic acid-lipid particle inaccordance with claim 1, wherein the interfering RNA is selected fromthe group consisting of siRNA, aiRNA, miRNA, Dicer-substrate dsRNA,shRNA, ssRNAi oligonucleotides, and combinations thereof.
 5. The nucleicacid-lipid particle in accordance with claim 1, wherein the interferingRNA is an siRNA.
 6. (canceled)
 7. The nucleic acid-lipid particle inaccordance with claim 5, wherein one or more of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides. 8.The nucleic acid-lipid particle in accordance with claim 7, wherein themodified nucleotides comprise 2′-O-methyl (2′OMe) nucleotides. 9.(canceled)
 10. The nucleic acid-lipid particle in accordance with claim7, wherein from about 20% to about 40% of the nucleotides in thedouble-stranded region comprise modified nucleotides. 11-13. (canceled)14. The nucleic acid-lipid particle in accordance with claim 5, whereinthe siRNA comprises an antisense strand comprising the followingsequence: 5′-UAUUCAGUGUGAUGACACU-3′. (SEQ ID NO: 13)


15. The nucleic acid-lipid particle in accordance with claim 14, furthercomprising a sense strand comprising the following sequence:5′-AGUGUCAUCACACUGAAUA-3′. (SEQ ID NO: 14)


16. The nucleic acid-lipid particle in accordance with claim 14, whereinthe siRNA comprises at least one 2′-O-methyl (2′OMe) nucleotide. 17-20.(canceled)
 21. The nucleic acid-lipid particle in accordance with claim5, wherein the siRNA comprises an antisense strand comprising thefollowing sequence: 5′-UAUUCAGUGUGAUGACACU-3′ (SEQ ID NO:15), whereinthe bolded and underlined nucleotides are 2′OMe nucleotides.
 22. Thenucleic acid-lipid particle in accordance with claim 21, furthercomprising a sense strand comprising the following sequence:5′-AGUGUCAUCACACUGAAUA-3′ (SEQ ID NO:16), wherein the bolded andunderlined nucleotides are 2′OMe nucleotides. 23-26. (canceled)
 27. Thenucleic acid-lipid particle in accordance with claim 22, wherein thesiRNA consists of the following sense and antisense strand sequences:5′-AGUGUCAUCACACUGAAUACC-3′ (SEQ ID NO:4) 3′-GUUCACAGUAGUGUGACUUAU-5′(SEQ ID NO:11) wherein the bolded and underlined nucleotides are 2′OMenucleotides.
 28. (canceled)
 29. The nucleic acid-lipid particle inaccordance with claim 1, wherein the cationic lipid has the followingstructure:

or salts thereof, wherein: R¹ and R² are either the same or differentand are independently optionally substituted C₁₂-C₂₄ alkyl, optionallysubstituted C₁₂-C₂₄ alkenyl, optionally substituted C₁₂-C₂₄ alkynyl, oroptionally substituted C₁₂-C₂₄ acyl; R³ and R⁴ are either the same ordifferent and are independently optionally substituted C₁-C₆ alkyl,optionally substituted C₂-C₆ alkenyl, or optionally substituted C₂-C₆alkynyl or R³ and R⁴ may join to form an optionally substitutedheterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosenfrom nitrogen and oxygen; R⁵ is either absent or is hydrogen or C₁-C₆alkyl to provide a quaternary amine; m, n, and p are either the same ordifferent and are independently either 0, 1 or 2, with the proviso thatm, n, and p are not simultaneously 0; Y and Z are either the same ordifferent and are independently O, S, or NH.
 30. The nucleic acid-lipidparticle in accordance with claim 29, wherein the cationic lipid is2,2-dilinoleyl-4-(2-dimethylaminoethyl)[1,3]-dioxolane (DLin-K-C2-DMA).31-36. (canceled)
 37. The nucleic acid-lipid particle in accordance withclaim 1, further comprising a conjugated lipid that inhibits aggregationof particles.
 38. The nucleic acid-lipid particle in accordance withclaim 37, wherein the conjugated lipid that inhibits aggregation ofparticles is a polyethyleneglycol (PEG)-lipid conjugate. 39-56.(canceled)
 57. The nucleic acid-lipid particle in accordance with claim38, wherein the nucleic acid-lipid particle comprises about 57.1 mol %cationic lipid, about 7.1 mol % phospholipid, about 34.3 mol %cholesterol or a derivative thereof, and about 1.4 mol % PEG-lipidconjugate.
 58. (canceled)
 59. A method for introducing an interferingRNA that silences APOB expression into a cell, the method comprising:contacting the cell with a nucleic acid-lipid particle in accordancewith claim
 1. 60-66. (canceled)
 67. A method for the in vivo delivery ofan interfering RNA that silences APOB expression, the method comprising:administering to a mammal a nucleic acid-lipid particle in accordancewith claim
 1. 68-76. (canceled)